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Sample records for puf9 regulates mrnas

  1. Yeast glycolytic mRNAs are differentially regulated.

    PubMed Central

    Moore, P A; Sagliocco, F A; Wood, R M; Brown, A J

    1991-01-01

    The regulation of glycolytic genes in response to carbon source in the yeast Saccharomyces cerevisiae has been studied. When the relative levels of each glycolytic mRNA were compared during exponential growth on glucose or lactate, the various glycolytic mRNAs were found to be induced to differing extents by glucose. No significant differences in the stabilities of the PFK2, PGK1, PYK1, or PDC1 mRNAs during growth on glucose or lactate were observed. PYK::lacZ and PGK::lacZ fusions were integrated independently into the yeast genome at the ura3 locus. The manner in which these fusions were differentially regulated in response to carbon source was similar to that of their respective wild-type loci. Therefore, the regulation of glycolytic mRNA levels is mediated at the transcriptional level. When the mRNAs are ordered with respect to the glycolytic pathway, two peaks of maximal induction are observed at phosphofructokinase and pyruvate kinase. These enzymes (i) catalyze the two essentially irreversible steps on the pathway, (ii) are the two glycolytic enzymes that are circumvented during gluconeogenesis and hence are specific to glycolysis, and (iii) are encoded by mRNAs that we have shown previously to be coregulated at the translational level in S. cerevisiae (P. A. Moore, A. J. Bettany, and A. J. P. Brown, NATO ASI Ser. Ser. H Cell Biol. 49:421-432, 1990). This differential regulation of glycolytic mRNA levels might therefore have a significant influence upon glycolytic flux in S. cerevisiae. Images PMID:1922048

  2. Cyclic AMP stabilizes a class of developmentally regulated Dictyostelium discoideum mRNAs.

    PubMed

    Mangiarotti, G; Ceccarelli, A; Lodish, H F

    The stability of mRNA is an important facet of the regulation of protein synthesis. In mammalian cells most mRNAs have long half-lives (5-15 hours) but a substantial fraction are much less stable. There are few examples where the stability of a particular mRNA or class of mRNAs is specifically affected by environmental or developmental stimuli. Certain hormones cause specific stabilization of mRNAs species and preferential mRNA stability is important in the accumulation of globin and myosin mRNAs during the terminal stages of erythropoesis or myogenesis, respectively. Disaggregation of Dictyostelium discoideum aggregates induces the specific destabilization of a large class of developmentally regulated mRNAs; thus, this system is an excellent one in which to determine how such controls are effected. Here we show that addition of cyclic AMP to disaggregated cells specifically prevents the destabilization of these mRNAs.

  3. Growth Hormone-Regulated mRNAs and miRNAs in Chicken Hepatocytes

    PubMed Central

    Wang, Huijuan; Shao, Fang; Yu, JianFeng; Jiang, Honglin; Han, Yaoping; Gong, Daoqing; Gu, Zhiliang

    2014-01-01

    Growth hormone (GH) is a key regulatory factor in animal growth, development and metabolism. Based on the expression level of the GH receptor, the chicken liver is a major target organ of GH, but the biological effects of GH on the chicken liver are not fully understood. In this work we identified mRNAs and miRNAs that are regulated by GH in primary hepatocytes from female chickens through RNA-seq, and analyzed the functional relevance of these mRNAs and miRNAs through GO enrichment analysis and miRNA target prediction. A total of 164 mRNAs were found to be differentially expressed between GH-treated and control chicken hepatocytes, of which 112 were up-regulated and 52 were down-regulated by GH. A total of 225 chicken miRNAs were identified by the RNA-Seq analysis. Among these miRNAs 16 were up-regulated and 1 miRNA was down-regulated by GH. The GH-regulated mRNAs were mainly involved in growth and metabolism. Most of the GH-upregulated or GH-downregulated miRNAs were predicted to target the GH-downregulated or GH-upregulated mRNAs, respectively, involved in lipid metabolism. This study reveals that GH regulates the expression of many mRNAs involved in metabolism in female chicken hepatocytes, which suggests that GH plays an important role in regulating liver metabolism in female chickens. The results of this study also support the hypothesis that GH regulates lipid metabolism in chicken liver in part by regulating the expression of miRNAs that target the mRNAs involved in lipid metabolism. PMID:25386791

  4. Synaptic microRNAs Coordinately Regulate Synaptic mRNAs: Perturbation by Chronic Alcohol Consumption.

    PubMed

    Most, Dana; Leiter, Courtney; Blednov, Yuri A; Harris, R Adron; Mayfield, R Dayne

    2016-01-01

    Local translation of mRNAs in the synapse has a major role in synaptic structure and function. Chronic alcohol use causes persistent changes in synaptic mRNA expression, possibly mediated by microRNAs localized in the synapse. We profiled the transcriptome of synaptoneurosomes (SN) obtained from the amygdala of mice that consumed 20% ethanol (alcohol) in a 30-day continuous two-bottle choice test to identify the microRNAs that target alcohol-induced mRNAs. SN are membrane vesicles containing pre- and post-synaptic compartments of neurons and astroglia and are a unique model for studying the synaptic transcriptome. We previously showed that chronic alcohol regulates mRNA expression in a coordinated manner. Here, we examine microRNAs and mRNAs from the same samples to define alcohol-responsive synaptic microRNAs and their predicted interactions with targeted mRNAs. The aim of the study was to identify the microRNA-mRNA synaptic interactions that are altered by alcohol. This was accomplished by comparing the effect of alcohol in SN and total homogenate preparations from the same samples. We used a combination of unbiased bioinformatic methods (differential expression, correlation, co-expression, microRNA-mRNA target prediction, co-targeting, and cell type-specific analyses) to identify key alcohol-sensitive microRNAs. Prediction analysis showed that a subset of alcohol-responsive microRNAs was predicted to target many alcohol-responsive mRNAs, providing a bidirectional analysis for identifying microRNA-mRNA interactions. We found microRNAs and mRNAs with overlapping patterns of expression that correlated with alcohol consumption. Cell type-specific analysis revealed that a significant number of alcohol-responsive mRNAs and microRNAs were unique to glutamate neurons and were predicted to target each other. Chronic alcohol consumption appears to perturb the coordinated microRNA regulation of mRNAs in SN, a mechanism that may explain the aberrations in synaptic

  5. Synaptic microRNAs Coordinately Regulate Synaptic mRNAs: Perturbation by Chronic Alcohol Consumption

    PubMed Central

    Most, Dana; Leiter, Courtney; Blednov, Yuri A; Harris, R Adron; Mayfield, R Dayne

    2016-01-01

    Local translation of mRNAs in the synapse has a major role in synaptic structure and function. Chronic alcohol use causes persistent changes in synaptic mRNA expression, possibly mediated by microRNAs localized in the synapse. We profiled the transcriptome of synaptoneurosomes (SN) obtained from the amygdala of mice that consumed 20% ethanol (alcohol) in a 30-day continuous two-bottle choice test to identify the microRNAs that target alcohol-induced mRNAs. SN are membrane vesicles containing pre- and post-synaptic compartments of neurons and astroglia and are a unique model for studying the synaptic transcriptome. We previously showed that chronic alcohol regulates mRNA expression in a coordinated manner. Here, we examine microRNAs and mRNAs from the same samples to define alcohol-responsive synaptic microRNAs and their predicted interactions with targeted mRNAs. The aim of the study was to identify the microRNA–mRNA synaptic interactions that are altered by alcohol. This was accomplished by comparing the effect of alcohol in SN and total homogenate preparations from the same samples. We used a combination of unbiased bioinformatic methods (differential expression, correlation, co-expression, microRNA-mRNA target prediction, co-targeting, and cell type-specific analyses) to identify key alcohol-sensitive microRNAs. Prediction analysis showed that a subset of alcohol-responsive microRNAs was predicted to target many alcohol-responsive mRNAs, providing a bidirectional analysis for identifying microRNA–mRNA interactions. We found microRNAs and mRNAs with overlapping patterns of expression that correlated with alcohol consumption. Cell type-specific analysis revealed that a significant number of alcohol-responsive mRNAs and microRNAs were unique to glutamate neurons and were predicted to target each other. Chronic alcohol consumption appears to perturb the coordinated microRNA regulation of mRNAs in SN, a mechanism that may explain the aberrations in synaptic

  6. Chromatin environment, transcriptional regulation, and splicing distinguish lincRNAs and mRNAs

    PubMed Central

    Melé, Marta; Mattioli, Kaia; Mallard, William; Shechner, David M.; Gerhardinger, Chiara; Rinn, John L.

    2017-01-01

    While long intergenic noncoding RNAs (lincRNAs) and mRNAs share similar biogenesis pathways, these transcript classes differ in many regards. LincRNAs are less evolutionarily conserved, less abundant, and more tissue-specific, suggesting that their pre- and post-transcriptional regulation is different from that of mRNAs. Here, we perform an in-depth characterization of the features that contribute to lincRNA regulation in multiple human cell lines. We find that lincRNA promoters are depleted of transcription factor (TF) binding sites, yet enriched for some specific factors such as GATA and FOS relative to mRNA promoters. Surprisingly, we find that H3K9me3—a histone modification typically associated with transcriptional repression—is more enriched at the promoters of active lincRNA loci than at those of active mRNAs. Moreover, H3K9me3-marked lincRNA genes are more tissue-specific. The most discriminant differences between lincRNAs and mRNAs involve splicing. LincRNAs are less efficiently spliced, which cannot be explained by differences in U1 binding or the density of exonic splicing enhancers but may be partially attributed to lower U2AF65 binding and weaker splicing-related motifs. Conversely, the stability of lincRNAs and mRNAs is similar, differing only with regard to the location of stabilizing protein binding sites. Finally, we find that certain transcriptional properties are correlated with higher evolutionary conservation in both DNA and RNA motifs and are enriched in lincRNAs that have been functionally characterized. PMID:27927715

  7. Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

    PubMed

    Sheets, Michael D; Fox, Catherine A; Dowdle, Megan E; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

    2017-01-01

    The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented

  8. Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos

    PubMed Central

    1990-01-01

    Xenopus mRNAs that potentially encode gap junction proteins in the oocyte and early embryo have been identified by low-stringency screening of cDNA libraries with cloned mammalian gap junction cDNAs. The levels of these mRNAs show strikingly different temporal regulation and tissue distribution. Using a nomenclature designed to stress important structural similarities of distinct gap junction gene products, the deduced polypeptides have been designated the Xenopus alpha 1 and alpha 2 gap junction proteins. The alpha 2 gap junction mRNA is a maternal transcript that disappears by the late gastrula stage. It is not detected in any organ of the adult except the ovary, and resides primarily, if not exclusively, in the oocytes and early embryos. The alpha 1 gap junction mRNA appears during organogenesis, and is detected in RNA from a wide variety of organs. It is also found in full-grown oocytes, but is rapidly degraded upon oocyte maturation, both in vivo and in vitro. The alpha 1 and alpha 2 mRNAs encode proteins with different degrees of amino acid sequence similarity to the predominant gap junction subunit of the mammalian heart (connexin 43). Together with our earlier report of a mid-embryonic (beta 1) gap junction mRNA, the results suggest that intercellular communication during oocyte growth and postfertilization development is a complex phenomenon involving the coordinated regulation of several genes. PMID:2155241

  9. Translational Regulation of Specific mRNAs Controls Feedback Inhibition and Survival during Macrophage Activation

    PubMed Central

    Schott, Johanna; Reitter, Sonja; Philipp, Janine; Haneke, Katharina; Schäfer, Heiner; Stoecklin, Georg

    2014-01-01

    For a rapid induction and efficient resolution of the inflammatory response, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level. The control of mRNA translation has emerged as an important determinant of protein levels, yet its role in macrophage activation is not well understood. We systematically analyzed the contribution of translational regulation to the early phase of the macrophage response by polysome fractionation from mouse macrophages stimulated with lipopolysaccharide (LPS). Individual mRNAs whose translation is specifically regulated during macrophage activation were identified by microarray analysis. Stimulation with LPS for 1 h caused translational activation of many feedback inhibitors of the inflammatory response including NF-κB inhibitors (Nfkbid, Nfkbiz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (Zfp36 and Zc3h12a). Our analysis showed that their translation is repressed in resting and de-repressed in activated macrophages. Quantification of mRNA levels at a high temporal resolution by RNASeq allowed us to define groups with different expression patterns. Thereby, we were able to distinguish mRNAs whose translation is actively regulated from mRNAs whose polysomal shifts are due to changes in mRNA levels. Active up-regulation of translation was associated with a higher content in AU-rich elements (AREs). For one example, Ier3 mRNA, we show that repression in resting cells as well as de-repression after stimulation depends on the ARE. Bone-marrow derived macrophages from Ier3 knockout mice showed reduced survival upon activation, indicating that IER3 induction protects macrophages from LPS-induced cell death. Taken together, our analysis reveals that translational control during macrophage activation is important for cellular survival as well as the expression of anti-inflammatory feedback inhibitors that promote the

  10. Extensive editing of mRNAs for the squid delayed rectifier K+ channel regulates subunit tetramerization.

    PubMed

    Rosenthal, Joshua J C; Bezanilla, Francisco

    2002-05-30

    We report the extensive editing of mRNAs that encode the classical delayed rectifier K+ channel (SqK(v)1.1A) in the squid giant axon. Using a quantitative RNA editing assay, 14 adenosine to guanine transitions were identified, and editing efficiency varied tremendously between positions. Interestingly, half of the sites are targeted to the T1 domain, important for subunit assembly. Other sites occur in the channel's transmembrane spans. The effects of editing on K+ channel function are elaborate. Edited codons affect channel gating, and several T1 sites regulate functional expression as well. In particular, the edit R87G, a phylogenetically conserved position, reduces expression close to 50-fold by regulating the channel's ability to form tetramers. These data suggest that RNA editing plays a dynamic role in regulating action potential repolarization in the giant axon.

  11. Basal Splicing Factors Regulate the Stability of Mature mRNAs in Trypanosomes*

    PubMed Central

    Gupta, Sachin Kumar; Carmi, Shai; Ben-Asher, Hiba Waldman; Tkacz, Itai Dov; Naboishchikov, Ilana; Michaeli, Shulamit

    2013-01-01

    Gene expression in trypanosomes is mainly regulated post-transcriptionally. Genes are transcribed as polycistronic mRNAs that are dissected by the concerted action of trans-splicing and polyadenylation. In trans-splicing, a common exon, the spliced leader, is added to all mRNAs from a small RNA. In this study, we examined by microarray analysis the transcriptome following RNAi silencing of the basal splicing factors U2AF65, SF1, and U2AF35. The transcriptome data revealed correlations between the affected genes and their splicing and polyadenylation signaling properties, suggesting that differential binding of these factors to pre-mRNA regulates trans-splicing and hence expression of specific genes. Surprisingly, all these factors were shown to affect not only splicing but also mRNA stability. Affinity purification of SF1 and U2AF35 complexes supported their role in mRNA stability. U2AF35 but not SF1 was shown to bind to ribosomes. To examine the role of splicing factors in mRNA stability, mutations were introduced into the polypyrimidine tract located in the 3′ UTR of a mini-gene, and the results demonstrate that U2AF65 binds to such a site and controls the mRNA stability. We propose that transcripts carrying splicing signals in their 3′ UTR bind the splicing factors and control their stability. PMID:23283975

  12. Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

    PubMed Central

    Becker, Jordan T.

    2017-01-01

    ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency

  13. mRNAs involved in copper homeostasis are regulated by the nonsense-mediated mRNA decay pathway depending on environmental conditions.

    PubMed

    Peccarelli, Megan; Scott, Taylor D; Steele, Megan; Kebaara, Bessie W

    2016-01-01

    The nonsense-mediated mRNA decay pathway (NMD) is an mRNA degradation pathway that degrades mRNAs that prematurely terminate translation. These mRNAs include mRNAs with premature termination codons as well as many natural mRNAs. In Saccharomyces cerevisiae a number of features have been shown to target natural mRNAs to NMD. However, the extent to which natural mRNAs from the same functional group are regulated by NMD and how environmental conditions influence this regulation is not known. Here, we examined mRNAs involved in copper homeostasis and are predicted to be sensitive to NMD. We found that the majority of these mRNAs have long 3'-UTRs that could target them for degradation by NMD. Analysis of one of these mRNAs, COX19, found that the long 3'-UTR contributes to regulation of this mRNA by NMD. Furthermore, we examined an additional mRNA, MAC1 under low copper conditions. We found that low copper growth conditions affect NMD sensitivity of the MAC1 mRNA demonstrating that sensitivity to NMD can be altered by environmental conditions. MAC1 is a copper sensitive transcription factor that regulates genes involved with high affinity copper transport. Our results expand our understanding of how NMD regulates mRNAs from the same functional group and how the environment influences this regulation.

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

    PubMed Central

    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. PMID:26564160

  15. Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia nigra

    PubMed Central

    2012-01-01

    Background Increased risk of schizophrenia in adolescent males indicates that a link between the development of dopamine-related psychopathology and testosterone-driven brain changes may exist. However, contradictions as to whether testosterone increases or decreases dopamine neurotransmission are found and most studies address this in adult animals. Testosterone-dependent actions in neurons are direct via activation of androgen receptors (AR) or indirect by conversion to 17β-estradiol and activation of estrogen receptors (ER). How midbrain dopamine neurons respond to sex steroids depends on the presence of sex steroid receptor(s) and the level of steroid conversion enzymes (aromatase and 5α-reductase). We investigated whether gonadectomy and sex steroid replacement could influence dopamine levels by changing tyrosine hydroxylase (TH) protein and mRNA and/or dopamine breakdown enzyme mRNA levels [catechol-O-methyl transferase (COMT) and monoamine oxygenase (MAO) A and B] in the adolescent male rat substantia nigra. We hypothesized that adolescent testosterone would regulate sex steroid signaling through regulation of ER and AR mRNAs and through modulation of aromatase and 5α-reductase mRNA levels. Results We find ERα and AR in midbrain dopamine neurons in adolescent male rats, indicating that dopamine neurons are poised to respond to circulating sex steroids. We report that androgens (T and DHT) increase TH protein and increase COMT, MAOA and MAOB mRNAs in the adolescent male rat substantia nigra. We report that all three sex steroids increase AR mRNA. Differential action on ER pathways, with ERα mRNA down-regulation and ERβ mRNA up-regulation by testosterone was found. 5α reductase-1 mRNA was increased by AR activation, and aromatase mRNA was decreased by gonadectomy. Conclusions We conclude that increased testosterone at adolescence can shift the balance of sex steroid signaling to favor androgenic responses through promoting conversion of T to DHT and

  16. shot regulates the microtubule reorganization required for localization of axis-determining mRNAs during oogenesis.

    PubMed

    Lee, Jiyeon; Lee, Sujung; Chen, Cheng; Shim, Hyeran; Kim-Ha, Jeongsil

    2016-02-01

    The Drosophila mid-oogenesis stages are notable as the time when most maternal mRNAs become localized at discrete regions of the oocyte. Microtubule rearrangement occurs during this period and is critical for the localization of axis-determining maternal mRNAs. We have identified shot as a key player in establishing the cytoskeletal arrangement required for the spatial localization of axis-determining maternal mRNAs. We also found that the spatial distribution of the Shot protein is regulated by its mRNA localization. Our results suggest that the RNA localization mechanism is used not only for restricted accumulation of patterning molecules but also for the microtubule organization that leads to the initial development of oocyte polarity.

  17. Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

    PubMed Central

    Hafner, Markus; Max, Klaas E.A.; Bandaru, Pradeep; Morozov, Pavel; Gerstberger, Stefanie; Brown, Miguel; Molina, Henrik; Tuschl, Thomas

    2013-01-01

    Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. PMID:23481595

  18. M-phase regulation of the recruitment of mRNAs onto polysomes using the CDK1/cyclin B inhibitor aminopurvalanol.

    PubMed

    Le Breton, Magali; Bellé, Robert; Cormier, Patrick; Mulner-Lorillon, Odile; Morales, Julia

    2003-07-11

    Translation under the control of the universal cell cycle regulator CDK1/cyclin B was investigated during the first cell cycle in sea urchin embryos. The CDK1/cyclin B inhibitor aminopurvalanol arrested embryos at the G2/M transition. Polysomal mRNAs were purified from control and arrested embryos, and screened for specific mRNA recruitment or release at M-phase by subtractive hybridization. The polysomal repartition of clones issued from this screen was analyzed. Three specific mRNAs were selectively recruited onto polysomes at M-phase. Conversely, two other specific mRNAs were released from polysomes. The isolation of these translationally regulated mRNAs gives now important tools for insights into the regulation of protein synthesis by the cell cycle regulator CDK1-cyclin B.

  19. CRM 1-mediated degradation and agonist-induced down-regulation of beta-adrenergic receptor mRNAs.

    PubMed

    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.

  20. Endogenous microRNAs in human microvascular endothelial cells regulate mRNAs encoded by hypertension-related genes.

    PubMed

    Kriegel, Alison J; Baker, Maria Angeles; Liu, Yong; Liu, Pengyuan; Cowley, Allen W; Liang, Mingyu

    2015-10-01

    The goal of this study was to systematically identify endogenous microRNAs (miRNAs) in endothelial cells that regulate mRNAs encoded by genes relevant to hypertension. Small RNA deep sequencing was performed in cultured human microvascular endothelial cells. Of the 50 most abundant miRNAs identified, 30 had predicted target mRNAs encoded by genes with known involvement in hypertension or blood pressure regulation. The cells were transfected with anti-miR oligonucleotides to inhibit each of the 30 miRNAs and the mRNA abundance of predicted targets was examined. Of 95 miRNA-target pairs examined, the target mRNAs were significantly upregulated in 35 pairs and paradoxically downregulated in 8 pairs. The result indicated significant suppression of the abundance of mRNA encoded by ADM by endogenous miR-181a-5p, ATP2B1 by the miR-27 family, FURIN by miR-125a-5p, FGF5 by the let-7 family, GOSR2 by miR-27a-3p, JAG1 by miR-21-5p, SH2B3 by miR-30a-5p, miR-98, miR-181a-5p, and the miR-125 family, TBX3 by the miR-92 family, ADRA1B by miR-22-3p, ADRA2A by miR-30a-5p and miR-30e-5p, ADRA2B by miR-30e-5p, ADRB1 by the let-7 family and miR-98, EDNRB by the miR-92 family, and NOX4 by the miR-92 family, miR-100-5p, and miR-99b-5p (n=3-9; P<0.05 versus scrambled anti-miR). Treatment with anti-miR-21 decreased blood pressure in mice fed a 4% NaCl diet. Inhibition of the miRNAs targeting NOX4 mRNA increased H2O2 release from endothelial cells. The findings indicate widespread, tonic control of mRNAs encoded by genes relevant to blood pressure regulation by endothelial miRNAs and provide a novel and uniquely informative basis for studying the role of miRNAs in hypertension.

  1. Differential Accumulation of Sunflower Tetraubiquitin mRNAs during Zygotic Embryogenesis and Developmental Regulation of Their Heat-Shock Response.

    PubMed Central

    Almoguera, C.; Coca, M. A.; Jordano, J.

    1995-01-01

    We have isolated and sequenced Ha UbiS, a cDNA for a dry-seed-stored mRNA that encodes tetraubiquitin. We have observed differential accumulation of tetraubiquitin mRNAs during sunflower (Helianthus annuus L.) zygotic embryogenesis. These mRNAs were up-regulated during late embryogenesis and reached higher prevalence in the dry seed, where they were found to be associated mainly with provascular tissue. UbiS mRNA, as confirmed by Rnase A protection experiments, accumulated also in response to heat shock, but only in leaves and later during postgerminative development. These novel observations demonstrate expression during seed maturation of specific plant polyubiquitin transcripts and developmental regulation of their heat-shock response. Using ubiquitin antibodies we also detected discrete, seed-specific proteins with distinct temporal expression patterns during zygotic embryogenesis. Some of these patterns were concurrent with UbiS mRNA accumulation in seeds. The most abundant ubiquitin-reacting proteins found in mature seeds were small (16-22 kD) and acidic (isoelectric points of 6.1-7.4). Possible functional implications for UbiS expression elicited from these observations are discussed. PMID:12228401

  2. A 5′ cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

    SciTech Connect

    Zhu, Deyu; Stumpf, Craig R.; Krahn, Joseph M.; Wickens, Marvin; Tanaka Hall, Traci M.

    2010-11-03

    A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the 'core' PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

  3. Registered report: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology.

    PubMed

    Cowley, Dale; Pandya, Kumar; Khan, Israr; Kerwin, John; Owen, Kate; Griner, Erin

    2015-09-03

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'A coding-independent function of gene and pseudogene mRNAs regulates tumour biology' by Poliseno et al. (2010), published in Nature in 2010. The key experiments to be replicated are reported in Figures 1D, 2F-H, and 4A. In these experiments, Poliseno and colleagues report microRNAs miR-19b and miR-20a transcriptionally suppress both PTEN and PTENP1 in prostate cancer cells (Figure 1D; Poliseno et al., 2010). Decreased expression of PTEN and/or PTENP1 resulted in downregulated PTEN protein levels (Figure 2H), downregulation of both mRNAs (Figure 2G), and increased tumor cell proliferation (Figure 2F; Poliseno et al., 2010). Furthermore, overexpression of the PTEN 3' UTR enhanced PTENP1 mRNA abundance limiting tumor cell proliferation, providing additional evidence for the co-regulation of PTEN and PTENP1 (Figure 4A; Poliseno et al., 2010). The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published in eLife.

  4. Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amoebae into flagellates

    SciTech Connect

    Lee, J.H.; Walsh, C.J.

    1988-06-01

    The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins ..cap alpha..- and BETA-tubulin and flagellar calmodulin, as well as a coordinately regulated poly(A)/sup +/ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)/sup +/ RNAs common to ameobae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in ameobae increased 3- to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flagellar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. gruberi amebae to form visible flagella.

  5. Role of 3′UTRs in the Translation of mRNAs Regulated by Oncogenic eIF4E—A Computational Inference

    PubMed Central

    Rajasekhar, Vinagolu K.; Larsson, Ola; Sonenberg, Nahum; Colburn, Nancy H.; Shapiro, Bruce A.

    2009-01-01

    Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3′UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3′UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5′UTR and microRNA binding at the 3′UTR. PMID:19290046

  6. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

    PubMed Central

    Soheilypour, M.; Mofrad, M. R. K.

    2016-01-01

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

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

    PubMed Central

    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

  8. The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway

    PubMed Central

    Coyle, John H.; Bor, Yeou-Cherng; Rekosh, David; Hammarskjold, Marie-Louise

    2011-01-01

    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. PMID:21613532

  9. Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis.

    PubMed

    Jensen, Liselotte E; Whitehead, Alexander S

    2003-08-01

    Two alternative splice variants of the interleukin-1 receptor accessory protein (IL-1RAcP) mRNA are known. Membrane-bound IL-1RAcP (mIL-1RAcP) promotes intracellular interleukin-1 (IL-1) signalling whereas soluble IL-1RAcP (sIL-1RAcP) is probably an inhibitor of IL-1 signalling. Here we establish that sIL-1RAcP mRNA levels increase 16-fold in response to phorbol esters in the human hepatoma cell line HepG2 via a mechanism that depends on de novo protein synthesis. Following exposure of cells to UV light, a potent inducer of apoptosis, mIL-1RAcP mRNA is rapidly down-regulated and a new steady-state level established briefly before a gradual return to pretreatment levels. Following treatment with staurosporine, also an inducer of apoptosis, mIL-1RAcP mRNA levels steadily decrease through 72 h, with little change in sIL-1RAcP mRNA levels. A novel alternative splice variant, sIL-1RAcP-beta, was identified. Its sequence indicates that sIL-1RAcP-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of IL-1RAcP mRNAs suggest important functions in regulating sensitivity to IL-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.

  10. Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells.

    PubMed

    Lee, Jerome E; Lee, Ju Youn; Wilusz, Jeffrey; Tian, Bin; Wilusz, Carol J

    2010-06-21

    Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific. We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1, consistent with the role of CUGBP1 as a

  11. Systematic Analysis of Cis-Elements in Unstable mRNAs Demonstrates that CUGBP1 Is a Key Regulator of mRNA Decay in Muscle Cells

    PubMed Central

    Wilusz, Jeffrey; Tian, Bin; Wilusz, Carol J.

    2010-01-01

    Background Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific. Principal Findings We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3′UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3′UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3′UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1, consistent

  12. Regulation of mRNAs encoding the steroidogenic acute regulatory protein and cholesterol side-chain cleavage enzyme in the elasmobranch interrenal gland.

    PubMed

    Evans, Andrew N; Nunez, B Scott

    2010-08-01

    The rate-limiting and regulated step in steroidogenesis, the conversion of cholesterol to pregnenolone, is facilitated by the steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage (P450scc). We have isolated cDNAs encoding StAR and P450scc from the Atlantic stingray, Dasyatis sabina, and characterized the steroidogenic activity of the encoded proteins using a heterologous expression system. Green monkey kidney (COS-1) cells cotransfected with D. sabina StAR and human P450scc/adrenodoxin reductase/adrenodoxin fusion (F2) constructs produced significantly more pregnenolone than cells transfected with the F2 construct alone. COS-1 cells transfected with a modified F2 construct (F2DS) in which human P450scc is replaced by D. sabina P450scc had higher rates than cells transfected with D. sabina P450scc alone. In other vertebrates, the stress peptide adrenocorticotropic hormone (ACTH) elicits its effects on corticosteroidogenesis in part through regulation of StAR and P450scc mRNAs. In vitro incubation of D. sabina interrenal tissue with porcine ACTH significantly increased intracellular cAMP and corticosteroid production. As demonstrated by quantitative PCR, ACTH also induced significant increases in mRNA abundance of both StAR and P450scc. Our results suggest that, as in higher vertebrates, chronic ACTH-induced glucocorticoid synthesis in elasmobranchs is mediated by regulation of primary steroidogenic mRNAs. This study is the first to demonstrate steroidogenic activity of an elasmobranch P450scc protein and express a composite elasmobranch steroidogenic pathway in a heterologous cell line. Also, the regulation of StAR and P450scc mRNAs has not previously been demonstrated in elasmobranch fishes. Copyright 2010 Elsevier Inc. All rights reserved.

  13. CELF4 Regulates Translation and Local Abundance of a Vast Set of mRNAs, Including Genes Associated with Regulation of Synaptic Function

    PubMed Central

    Sun, Wenzhi; Mahaffey, Connie L.; Curk, Tomaž; Rot, Gregor; Ule, Jernej; Frankel, Wayne N.

    2012-01-01

    RNA–binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA–binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in excitatory neurons, including large pyramidal cells of the cerebral cortex and hippocampus, and it regulates excitatory but not inhibitory neurotransmission. We examined mechanisms underlying neuronal hyperexcitability in Celf4 mutants by identifying CELF4 target mRNAs and assessing their fate in the absence of CELF4 in view of their known functions. CELF4 binds to at least 15%–20% of the transcriptome, with striking specificity for the mRNA 3′ untranslated region. CELF4 mRNA targets encode a variety of proteins, many of which are well established in neuron development and function. While the overall abundance of these mRNA targets is often dysregulated in Celf4 deficient mice, the actual expression changes are modest at the steady-state level. In contrast, by examining the transcriptome of polysome fractions and the mRNA distribution along the neuronal cell body-neuropil axis, we found that CELF4 is critical for maintaining mRNA stability and availability for translation. Among biological processes associated with CELF4 targets that accumulate in neuropil of mutants, regulation of synaptic plasticity and transmission are the most prominent. Together with a related study of the impact of CELF4 loss on sodium channel Nav1.6 function, we suggest that CELF4 deficiency leads to abnormal neuronal function by combining a specific effect on neuronal excitation with a general impairment of synaptic transmission. These results also expand our understanding of the vital roles RNA–binding proteins play in regulating and shaping the activity of neural circuits. PMID:23209433

  14. Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3'-untranslated region of their mRNAs.

    PubMed

    Roca, Francisco J; Cayuela, María L; Secombes, Chris J; Meseguer, José; Mulero, Victoriano

    2007-01-01

    The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.

  15. Repeated Exposure to D-Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

    PubMed Central

    Biever, Anne; Boubaker-Vitre, Jihane; Cutando, Laura; Gracia-Rubio, Irene; Costa-Mattioli, Mauro; Puighermanal, Emma; Valjent, Emmanuel

    2017-01-01

    Repeated psychostimulant exposure induces persistent gene expression modifications that contribute to enduring changes in striatal GABAergic spiny projecting neurons (SPNs). However, it remains unclear whether changes in the control of mRNA translation are required for the establishment of these durable modifications. Here we report that repeated exposure to D-amphetamine decreases global striatal mRNA translation. This effect is paralleled by an enhanced phosphorylation of the translation factors, eIF2α and eEF2, and by the concomitant increased translation of a subset of mRNAs, among which the mRNA encoding for the activity regulated cytoskeleton-associated protein, also known as activity regulated gene 3.1 (Arc/Arg3.1). The enrichment of Arc/Arg3.1 mRNA in the polysomal fraction is accompanied by a robust increase of Arc/Arg3.1 protein levels within the striatum. Immunofluorescence analysis revealed that this increase occurred preferentially in D1R-expressing SPNs localized in striosome compartments. Our results suggest that the decreased global protein synthesis following repeated exposure to D-amphetamine favors the translation of a specific subset of mRNAs in the striatum. PMID:28119566

  16. Binding of LARP6 to the conserved 5' stem-loop regulates translation of mRNAs encoding type I collagen.

    PubMed

    Cai, Le; Fritz, Dillon; Stefanovic, Lela; Stefanovic, Branko

    2010-01-15

    Type I collagen is the most abundant protein in the human body, produced by folding of two alpha1(I) polypeptides and one alpha2(I) polypeptide into the triple helix. A conserved stem-loop structure is found in the 5' untranslated region of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein that binds the collagen 5' stem-loop in a sequence-specific manner. LARP6 has a distinctive bipartite RNA binding domain not found in other members of the La superfamily. LARP6 interacts with the two single-stranded regions of the 5' stem-loop. The K(d) for binding of LARP6 to the 5' stem-loop is 1.4 nM. LARP6 binds the 5' stem-loop in both the nucleus and the cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes; however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using collagen-GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but only when the reporter was encoded by mRNA with the 5' stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5' stem-loop, or in the absence of LARP6, it accumulated diffusely throughout the endoplasmic reticulum. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulate that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer. Copyright 2009 Elsevier Ltd. All rights reserved.

  17. The expression of insulin and insulin receptor mRNAs is regulated by nutritional state and glucose in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Caruso, Michael A; Sheridan, Mark A

    2012-01-15

    Many species of fish, including rainbow trout, possess multiple INS- and IR-encoding mRNAs. In this study, rainbow trout (Oncorhynchus mykiss) were used as a model to study the regulation of INS (INS1, INS2) and IR (IR1, IR2, IR3, and IR4) mRNA expression by nutritional state and glucose. In the nutritional state study, fish were either fed continuously, fasted (4 or 6 weeks), or fasted 4 weeks, then refed for 2 weeks. Nutritional state regulated INS and IR mRNA expression in a subtype- and tissue-specific manner. A 4-week fast reduced INS1 expression in endocrine pancreas (Brockmann body) and of INS1 and INS2 in brain, whereas a 6-week fast reduced the expression of both INS1 and INS2 in pancreas but only of INS1 in brain. Refeeding only restored INS2 levels in pancreas. In adipose tissue, by contrast, a 4-week fast increased INS1 expression, and a 6-week fast increased the expression of both INS1 and INS2. Nutritional state also modulated the pattern of IR mRNA expression. Fasting for 4 weeks resulted in no significant change in IR expression. Prolonged fasting (6 weeks) increased the expression of IR4 mRNA in the pancreas, adipose tissue, cardiac muscle, and gill; however, fasting decreased expression of IR3 mRNA in liver. Refeeding restored fasting-associated increases in IR4 expression in pancreas, adipose tissue, cardiac muscle, and gill, but had no effect on the fasting-associated decrease in IR3 expression in liver. Glucose differentially regulated the expression of INS and IR mRNAs in Brockmann bodies and liver pieces incubated in vitro, respectively. Low glucose (1 mM) reduced pancreatic expression of both INS1 and INS2 mRNAs compared to levels observed at 4 or 10 mM glucose. In the liver, IR1 and IR2 mRNA expression was insensitive to glucose concentration, whereas expression of IR3 and IR4 was attenuated at 1 and 10 mM compared to 4 mM glucose. These findings indicate that the pattern of INS and IR expression in selected tissues is regulated by

  18. The novel poly(A) polymerase Star-PAP is a signal-regulated switch at the 3'-end of mRNAs.

    PubMed

    Li, Weimin; Laishram, Rakesh S; Anderson, Richard A

    2013-01-01

    The mRNA 3'-untranslated region (3'-UTR) modulates message stability, transport, intracellular location and translation. We have discovered a novel nuclear poly(A) polymerase termed Star-PAP (nuclear speckle targeted PIPKIα regulated-poly(A) polymerase) that couples with the transcriptional machinery and is regulated by the phosphoinositide lipid messenger phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)), the central lipid in phosphoinositide signaling. PI4,5P(2) is generated primarily by type I phosphatidylinositol phosphate kinases (PIPKI). Phosphoinositides are present in the nucleus including at nuclear speckles compartments separate from known membrane structures. PIPKs regulate cellular functions by interacting with PI4,5P(2) effectors where PIPKs generate PI4,5P(2) that then modulates the activity of the associated effectors. Nuclear PIPKIα interacts with and regulates Star-PAP, and PI4,5P(2) specifically activates Star-PAP in a gene- and signaling-dependent manner. Importantly, other select signaling molecules integrated into the Star-PAP complex seem to regulate Star-PAP activities and processivities toward RNA substrates, and unique sequence elements around the Star-PAP binding sites within the 3'-UTR of target genes contribute to Star-PAP specificity for processing. Therefore, Star-PAP and its regulatory molecules form a signaling nexus at the 3'-end of target mRNAs to control the expression of select group of genes including the ones involved in stress responses.

  19. Maternal mRNAs are regulated by diverse P body–related mRNP granules during early Caenorhabditis elegans development

    PubMed Central

    Noble, Scott L.; Allen, Brittany L.; Goh, Lai Kuan; Nordick, Kristen; Evans, Thomas C.

    2008-01-01

    Processing bodies (P bodies) are conserved mRNA–protein (mRNP) granules that are thought to be cytoplasmic centers for mRNA repression and degradation. However, their specific functions in vivo remain poorly understood. We find that repressed maternal mRNAs and their regulators localize to P body–like mRNP granules in the Caenorhabditis elegans germ line. Surprisingly, several distinct types of regulated granules form during oocyte and embryo development. 3′ untranslated region elements direct mRNA targeting to one of these granule classes. The P body factor CAR-1/Rap55 promotes association of repressed mRNA with granules and contributes to repression of Notch/glp-1 mRNA. However, CAR-1 controls Notch/glp-1 only during late oogenesis, where it functions with the RNA-binding regulators PUF-5, PUF-6, and PUF-7. The P body protein CGH-1/Rck/Dhh1 differs from CAR-1 in control of granule morphology and promotes mRNP stability in arrested oocytes. Therefore, a system of diverse and regulated RNP granules elicits stage-specific functions that ensure proper mRNA control during early development. PMID:18695046

  20. Regulation of Hypoxia-Inducible mRNAs by the von Hippel-Lindau Tumor Suppressor Protein Requires Binding to Complexes Containing Elongins B/C and Cul2

    PubMed Central

    Lonergan, Kim M.; Iliopoulos, Othon; Ohh, Michael; Kamura, Takumi; Conaway, Ronald C.; Conaway, Joan Weliky; Kaelin, William G.

    1998-01-01

    The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis. PMID:9447969

  1. An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates*

    PubMed Central

    O'Day, Elizabeth; Le, Minh T. N.; Imai, Shunsuke; Tan, Shen Mynn; Kirchner, Rory; Arthanari, Haribabu; Hofmann, Oliver; Wagner, Gerhard; Lieberman, Judy

    2015-01-01

    Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function. PMID:26045559

  2. An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates.

    PubMed

    O'Day, Elizabeth; Le, Minh T N; Imai, Shunsuke; Tan, Shen Mynn; Kirchner, Rory; Arthanari, Haribabu; Hofmann, Oliver; Wagner, Gerhard; Lieberman, Judy

    2015-07-17

    Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Differential expression profile analysis of lncRNA UCA1α regulated mRNAs in bladder cancer.

    PubMed

    Wang, Yu; Zhang, Hong; Li, Xu; Chen, Wei

    2017-08-16

    Urothelial carcinoma associated 1α (UCA1α) is a novel long non-coding RNA (lncRNA) that regulates bladder cancer proliferation, migration, and invasion. The target genes of UCA1α have, however, not been identified. To address this, a pCDNA3.1(+)-UCA1α over-expression vector was transfected into UM-UC-2 bladder cancer cells. Genes differentially expressed between pCDNA3.1(+)-UCA1α and pCDNA3.1(+) transfected cell were then detected by microarray and bioinformatics analysis. A total of 71 differentially expressed genes were identified, including 52 up-regulated genes and 19 down-regulated genes. As expected, the lncRNA UCA1α expression level was significantly increased when compared to that of pCDNA3.1(+) transfected cells. The five most significantly up-regulated and five most significantly down-regulated genes were selected, and their expression levels were also assessed by real time quantitative polymerase chain reaction and Western blot. The mRNA and protein expression levels of FOXI3 and GSTA3 were found to be significantly increased, and those of MED18 and TEX101 were found to be significantly decreased. Gene ontology (GO) clustering identified several significant biological processes, cellular components, and molecular functions, associated with lncRNA UCA1α over-expression. The differentially expressed genes were involved in several significant pathways as shown by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway clustering. Cell proliferation activity was significantly increased following overexpression of lncRNA UCA1α increasing over culture time. The present study identifies, for the first time, potential target genes for lncRNA UCA1α in bladder cancer, and provides a significant reference for studying the role of lncRNA UCA1α in bladder cancer. © 2017 Wiley Periodicals, Inc.

  4. The Novel Poly(A) Polymerase Star-PAP is a Signal-Regulated Switch at the 3′-end of mRNAs

    PubMed Central

    Li, Weimin; Laishram, Rakesh S.; Anderson, Richard A.

    2013-01-01

    The mRNA 3′-untranslated region (3′-UTR) modulates message stability, transport, intracellular location and translation. We have discovered a novel nuclear poly(A) polymerase termed Star-PAP (nuclear speckle targeted PIPKIα regulated-poly(A) polymerase) that couples with the transcriptional machinery and is regulated by the phosphoinositide lipid messenger phosphatidylinositol-4,5-bisphosphate (PI4,5P2), the central lipid in phosphoinositide signaling. PI4,5P2 is generated primarily by type I phosphatidylinositol phosphate kinases (PIPKI). Phosphoinositides are present in the nucleus including at nuclear speckles compartments separate from known membrane structures. PIPKs regulate cellular functions by interacting with PI4,5P2 effectors where PIPKs generate PI4,5P2 that then modulates the activity of the associated effectors. Nuclear PIPKIα interacts with and regulates Star-PAP, and PI4,5P2 specifically activates Star-PAP in a gene- and signaling-dependent manner. Importantly, other select signaling molecules integrated into the Star-PAP complex seem to regulate Star-PAP activities and processivities toward RNA substrates, and unique sequence elements around the Star-PAP binding sites within the 3′-UTR of target genes contribute to Star-PAP specificity for processing. Therefore, Star-PAP and its regulatory molecules form a signaling nexus at the 3′-end of target mRNAs to control the expression of select group of genes including the ones involved in stress responses. PMID:23306079

  5. The 2-5A/RNase L/RNase L inhibitor (RLI) [correction of (RNI)] pathway regulates mitochondrial mRNAs stability in interferon alpha-treated H9 cells.

    PubMed

    Le Roy, F; Bisbal, C; Silhol, M; Martinand, C; Lebleu, B; Salehzada, T

    2001-12-21

    Interferon alpha (IFNalpha) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNalpha exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNalpha is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNalpha and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNalpha. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNalpha treatment in these transfectants. These results demonstrate that IFNalpha exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.

  6. RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3′ UTRs

    PubMed Central

    Di Giammartino, Dafne Campigli; Li, Wencheng; Ogami, Koichi; Yashinskie, Jossie J.; Hoque, Mainul; Tian, Bin

    2014-01-01

    Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3′ processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN (“domain with no name”), that is required for 3′ processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3′ processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3′ untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3′ processing, especially of RNAs with AU-rich 3′ UTRs. PMID:25319826

  7. Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

    SciTech Connect

    Grigoriev, Igor; Nicolas, Francisco; Moxon, Simon; Haro, Juan de; Calo, Silvia; Torres-Martinez, Santiago; Moulton, Vincent; Ruiz-Vazquez, Rosa; Dalmay, Tamas

    2011-09-01

    Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi

  8. The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

    PubMed Central

    Rinaldi, Arlie J.; Lund, Paul E.; Blanco, Mario R.; Walter, Nils G.

    2016-01-01

    In response to intracellular signals in Gram-negative bacteria, translational riboswitches—commonly embedded in messenger RNAs (mRNAs)—regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by ‘bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation. PMID:26781350

  9. The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells.

    PubMed

    Chennathukuzhi, Vargheese; Morales, Carlos R; El-Alfy, Mohamed; Hecht, Norman B

    2003-12-23

    Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of the human protein Translin, is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport, stabilization, and storage. Targeted inactivation of TB-RBP leads to abnormalities in fertility and behavior. A testis-enriched kinesin KIF17b coimmunoprecipitates with TB-RBP in a RNA-protein complex containing specific cAMP-responsive element modulator (CREM)-regulated mRNAs. The specificity of this interaction is confirmed by in vivo RNA-protein crosslinking and transfections of hippocampal neurons. Combining in situ hybridization and immunohistochemistry at the electron microscope level, a temporally sequential dissociation of KIF17b and TB-RBP from specific mRNAs is detected with TB-RBP release coincident with the time of mRNA translation, indicating a separation of the processes of transport and translation. We conclude that KIF17b serves as a molecular motor component of a TB-RBP-mouse ribonucleoprotein complex transporting a group of specific CREM-regulated mRNAs in mammalian male postmeiotic germ cells. Because KIF17b has been reported to control CREM-dependent transcription in male germ cells by regulating the intracellular location of the transcriptional coactivator activator of CREM in testis, this indicates that one kinesin links the processes of transcription and transport of specific mRNAs in mammalian male germ cells.

  10. Joint Profiling of miRNAs and mRNAs Reveals miRNA Mediated Gene Regulation in the Göttingen Minipig Obesity Model

    PubMed Central

    Alkan, Ferhat; Keinicke, Helle; Jacobsen, Mette J.; Gorodkin, Jan; Fredholm, Merete; Cirera, Susanna

    2016-01-01

    Obesity and its comorbidities are an increasing challenge for both affected individuals and health care systems, worldwide. In obese individuals, perturbation of expression of both protein-coding genes and microRNAs (miRNA) are seen in obesity-relevant tissues (i.e. adipose tissue, liver and skeletal muscle). miRNAs are small non-coding RNA molecules which have important regulatory roles in a wide range of biological processes, including obesity. Rodents are widely used animal models for human diseases including obesity. However, not all research is applicable for human health or diseases. In contrast, pigs are emerging as an excellent animal model for obesity studies, due to their similarities in their metabolism, their digestive tract and their genetics, when compared to humans. The Göttingen minipig is a small sized easy-to-handle pig breed which has been extensively used for modeling human obesity, due to its capacity to develop severe obesity when fed ad libitum. The aim of this study was to identify differentially expressed of protein-coding genes and miRNAs in a Göttingen minipig obesity model. Liver, skeletal muscle and abdominal adipose tissue were sampled from 7 lean and 7 obese minipigs. Differential gene expression was investigated using high-throughput quantitative real-time PCR (qPCR) on 90 mRNAs and 72 miRNAs. The results revealed de-regulation of several obesity and inflammation-relevant protein-coding genes and miRNAs in all tissues examined. Many genes that are known to be de-regulated in obese humans were confirmed in the obese minipigs and several of these genes have target sites for miRNAs expressed in the opposing direction of the gene, confirming miRNA-mediated regulation in obesity. These results confirm the translational value of the pig for human obesity studies. PMID:27902747

  11. Isolation of cucumber CsARF cDNAs and expression of the corresponding mRNAs during gravity-regulated morphogenesis of cucumber seedlings

    NASA Astrophysics Data System (ADS)

    Saito, Y.; Yamasaki, S.; Fujii, N.; Hagen, G.; Guilfoyle, T.; Takahashi, H.

    Cucumber seedlings grown in a horizontal position develop a protuberance called peg on the lower side of the transition zone between the hypocotyl and the root. We have suggested that peg formation on the upper side of the gravistimulated transition zone is suppressed because cucumber seedlings grown in a vertical position or microgravity symmetrically develop two pegs on the transition zone. Plant hormone, auxin, is considered to play a crucial role in the gravity-regulated formation of peg. We have shown that the mRNAs of auxin-inducible genes (CsIAAs) isolated from cucumber accumulate more abundantly in the lower side of the transition zone than in the upper side when peg formation initiates. To reveal the mechanism of transcriptional regulation by auxin for peg formation, we isolated five cDNAs of Auxin Response Factors (ARFs) from cucumber and compared their mRNA accumulation with those of CsIAA1 and CsIAA2. The tissue specificity of mRNA accumulation of CsARF2 was similar to those of CsIAA1 and CsIAA2. The structural character of CsARF2 predicts it is transcriptional activator. These results suggest that CsARF2 may be involved in activation of the transcription of auxin-inducible genes including CsIAA1 for peg formation. Because mRNA accumulation of five CsARFs, including CsARF2, were affected by neither gravity nor auxin, transcriptional activity of CsARF2 may be regulated at post-transcriptional level to induce asymmetric mRNA accumulation of auxin-inducible genes in the transition zone.

  12. Joint Profiling of miRNAs and mRNAs Reveals miRNA Mediated Gene Regulation in the Göttingen Minipig Obesity Model.

    PubMed

    Mentzel, Caroline M Junker; Alkan, Ferhat; Keinicke, Helle; Jacobsen, Mette J; Gorodkin, Jan; Fredholm, Merete; Cirera, Susanna

    2016-01-01

    Obesity and its comorbidities are an increasing challenge for both affected individuals and health care systems, worldwide. In obese individuals, perturbation of expression of both protein-coding genes and microRNAs (miRNA) are seen in obesity-relevant tissues (i.e. adipose tissue, liver and skeletal muscle). miRNAs are small non-coding RNA molecules which have important regulatory roles in a wide range of biological processes, including obesity. Rodents are widely used animal models for human diseases including obesity. However, not all research is applicable for human health or diseases. In contrast, pigs are emerging as an excellent animal model for obesity studies, due to their similarities in their metabolism, their digestive tract and their genetics, when compared to humans. The Göttingen minipig is a small sized easy-to-handle pig breed which has been extensively used for modeling human obesity, due to its capacity to develop severe obesity when fed ad libitum. The aim of this study was to identify differentially expressed of protein-coding genes and miRNAs in a Göttingen minipig obesity model. Liver, skeletal muscle and abdominal adipose tissue were sampled from 7 lean and 7 obese minipigs. Differential gene expression was investigated using high-throughput quantitative real-time PCR (qPCR) on 90 mRNAs and 72 miRNAs. The results revealed de-regulation of several obesity and inflammation-relevant protein-coding genes and miRNAs in all tissues examined. Many genes that are known to be de-regulated in obese humans were confirmed in the obese minipigs and several of these genes have target sites for miRNAs expressed in the opposing direction of the gene, confirming miRNA-mediated regulation in obesity. These results confirm the translational value of the pig for human obesity studies.

  13. Specific Soybean Lipoxygenases Localize to Discrete Subcellular Compartments and Their mRNAs Are Differentially Regulated by Source-Sink Status1

    PubMed Central

    Stephenson, Lowry C.; Bunker, Thomas W.; Dubbs, Wesley E.; Grimes, Howard D.

    1998-01-01

    Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA–vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B). PMID:9501125

  14. Translation Regulation and RNA Granule Formation after Heat Shock of Procyclic Form Trypanosoma brucei: Many Heat-Induced mRNAs Are also Increased during Differentiation to Mammalian-Infective Forms.

    PubMed

    Minia, Igor; Merce, Clementine; Terrao, Monica; Clayton, Christine

    2016-09-01

    African trypanosome procyclic forms multiply in the midgut of tsetse flies, and are routinely cultured at 27°C. Heat shocks of 37°C and above result in general inhibition of translation, and severe heat shock (41°C) results in sequestration of mRNA in granules. The mRNAs that are bound by the zinc-finger protein ZC3H11, including those encoding refolding chaperones, escape heat-induced translation inhibition. At 27°C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37°C-41°C, the ZC3H11 mRNA moves into the polysomal fraction. To investigate the scope and specificities of heat-shock translational regulation and granule formation, we analysed the distributions of mRNAs on polysomes at 27°C and after 1 hour at 39°C, and the mRNA content of 41°C heat shock granules. We found that mRNAs that bind to ZC3H11 remained in polysomes at 39°C and were protected from sequestration in granules at 41°C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. 70 mRNAs moved towards the polysomal fraction after the 39°C heat shock, and 260 increased in relative abundance. Surprisingly, many of these mRNAs are also increased when trypanosomes migrate to the tsetse salivary glands. It therefore seems possible that in the wild, temperature changes due to diurnal variations and periodic intake of warm blood might influence the efficiency with which procyclic forms develop into mammalian-infective forms.

  15. Translation Regulation and RNA Granule Formation after Heat Shock of Procyclic Form Trypanosoma brucei: Many Heat-Induced mRNAs Are also Increased during Differentiation to Mammalian-Infective Forms

    PubMed Central

    Minia, Igor; Merce, Clementine; Terrao, Monica

    2016-01-01

    African trypanosome procyclic forms multiply in the midgut of tsetse flies, and are routinely cultured at 27°C. Heat shocks of 37°C and above result in general inhibition of translation, and severe heat shock (41°C) results in sequestration of mRNA in granules. The mRNAs that are bound by the zinc-finger protein ZC3H11, including those encoding refolding chaperones, escape heat-induced translation inhibition. At 27°C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37°C—41°C, the ZC3H11 mRNA moves into the polysomal fraction. To investigate the scope and specificities of heat-shock translational regulation and granule formation, we analysed the distributions of mRNAs on polysomes at 27°C and after 1 hour at 39°C, and the mRNA content of 41°C heat shock granules. We found that mRNAs that bind to ZC3H11 remained in polysomes at 39°C and were protected from sequestration in granules at 41°C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. 70 mRNAs moved towards the polysomal fraction after the 39°C heat shock, and 260 increased in relative abundance. Surprisingly, many of these mRNAs are also increased when trypanosomes migrate to the tsetse salivary glands. It therefore seems possible that in the wild, temperature changes due to diurnal variations and periodic intake of warm blood might influence the efficiency with which procyclic forms develop into mammalian-infective forms. PMID:27606618

  16. The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts

    PubMed Central

    Russo, Joseph; Lee, Jerome E.; López, Carolina M.; Anderson, John; Nguyen, Thuy-mi P.; Heck, Adam M.; Wilusz, Jeffrey

    2017-01-01

    We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion. PMID:28129347

  17. Ribosome Traffic on mRNAs Maps to Gene Ontology: Genome-wide Quantification of Translation Initiation Rates and Polysome Size Regulation

    PubMed Central

    Ciandrini, Luca

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function. PMID:23382661

  18. Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.

    PubMed

    Ciandrini, Luca; Stansfield, Ian; Romano, M Carmen

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function.

  19. Making sense of antisense: seemingly noncoding RNAs antisense to the master regulator of Kaposi's sarcoma-associated herpesvirus lytic replication do not regulate that transcript but serve as mRNAs encoding small peptides.

    PubMed

    Xu, Yiyang; Ganem, Don

    2010-06-01

    The mammalian transcriptome is studded with putative noncoding RNAs, many of which are antisense to known open reading frames (ORFs). Roles in the regulation of their complementary mRNAs are often imputed to these antisense transcripts, but few have been experimentally examined, and such functions remain largely conjectural. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two transcripts that lack obvious ORFs and are complementary to the gene (RTA) encoding the master regulator of the latent/lytic switch. Here, we show that, contrary to expectation, these RNAs do not regulate RTA expression. Rather, they are found on polysomes, and genetic analysis indicates that translational initiation occurs at several AUG codons in the RNA, leading to the presumptive synthesis of peptides of 17 to 48 amino acids. These findings underscore the need for circumspection in the computational assessment of coding potential and raise the possibility that the mammalian proteome may contain many previously unsuspected peptides generated from seemingly noncoding RNAs, some of which could have important biological functions. Irrespective of their function, such peptides could also contribute substantially to the repertoire of T cell epitopes generated in both uninfected and infected cells.

  20. Neuritogenesis induced by vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and peptide histidine methionine in SH-SY5y cells is associated with regulated expression of cytoskeleton mRNAs and proteins.

    PubMed

    Héraud, Céline; Hilairet, Sandrine; Muller, Jean-Marc; Leterrier, Jean-François; Chadéneau, Corinne

    2004-02-01

    Vasoactive intestinal peptide (VIP) and the related peptides pituitary adenylate cyclase-activating polypeptide (PACAP) and peptide histidine methionine (PHM) are known to regulate proliferation and/or differentiation in normal and tumoral cells. In this study, neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium was induced by VIP, PACAP, and PHM. The establishment of this process was followed by the quantification of neurite length and branching and the expression of neurofilament mRNAs, neurofilament proteins, and other cytoskeletal protein markers of neuronal differentiation: neuron-specific MAPs and beta-tubulin III. Neurite length and branching and the expression of most markers tested were increased by VIP and PACAP in a similar, although slightly different, fashion. In contrast, neuritic elongation induced by PHM was correlated with neither an increase in branching or neurofilament mRNAs nor a clear change in the expression of cytoskeleton proteins, with the exception of the stimulation by PHM of doublecortin, a microtubule-associated marker of migrating neuroblasts. These findings are the first evidence from a human neuron-like cell line for 1) a direct regulation of the metabolism of neurofilaments by VIP and PACAP and 2) the induction by PHM of neuritic processes of an apparent immature character. Copyright 2003 Wiley-Liss, Inc.

  1. Regulation of miR-29b-1/a transcription and identification of target mRNAs in CHO-K1 cells.

    PubMed

    Muluhngwi, Penn; Richardson, Kirsten; Napier, Joshua; Rouchka, Eric C; Mott, Justin L; Klinge, Carolyn M

    2017-03-15

    miR-29b and miR-29a transcript levels were reported to increase in exponentially growing CHO-K1 cells. Here, we examine the regulation of miR-29b-1/a in CHO-K1 cells. We observed that 4-hydroxytamoxifen (4-OHT) increased pri-miR-29b-1 and pri-miR-29a transcription in CHO-K1 cells by activating endogenous estrogen receptor α (ERα). DICER, an established, bona fide target of miR-29b-1/a, was shown to be regulated by 4-OHT in CHO-K1 cells. We showed that miR-29b-1 and miR-29a serve a repressive role in cell proliferation, migration, invasion, and colony formation in CHO-K1 cells. To identify other targets of miR-29b-1 and miR-29a, RNA sequencing was performed by transfecting cells with anti-miR-29a, which inhibits both miR-29a and miR-29b-1, pre-miR-29b-1, and/or pre-miR-29a. In silico network analysis in MetaCore™ identified common and unique putative gene targets of miR-29b-1 and miR-29a. Pathway analysis of identified putative miR-29 targets were related to cell adhesion, cytoskeletal remodeling, and development. Further inquiry revealed regulation of pathways mediating responses to growth factor stimulus and cell cycle regulation.

  2. A snoRNA modulates mRNA 3′ end processing and regulates the expression of a subset of mRNAs

    PubMed Central

    Huang, Chunliu; Shi, Junjie; Guo, Yibin; Huang, Weijun; Huang, Shanshan; Ming, Siqi; Wu, Xingui; Zhang, Rui; Ding, Junjun; Zhao, Wei; Jia, Jie; Huang, Xi; Xiang, Andy Peng

    2017-01-01

    Abstract mRNA 3′ end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3′ processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3′ processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3′ processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1–RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3′ processing.

  3. Drosha, DGCR8, and Dicer mRNAs are down-regulated in human cells infected with dengue virus 4, and play a role in viral pathogenesis.

    PubMed

    Casseb, S M M; Simith, D B; Melo, K F L; Mendonça, M H; Santos, A C M; Carvalho, V L; Cruz, A C R; Vasconcelos, P F C

    2016-05-09

    Dengue virus (DENV) and its four serotypes (DENV1-4) belong to the Flavivirus genus of the Flaviviridae family. DENV infection is a life-threatening disease, which results in up to 20,000 deaths each year. Viruses have been shown to encode trans-regulatory small RNAs, or microRNAs (miRNAs), which bind to messenger RNA and negatively regulate host or viral gene expression. During DENV infections, miRNAs interact with proteins in the RNAi pathway, and are processed by ribonucleases such as Dicer and Drosha. This study aims to investigate Drosha, DGCR8, and Dicer expression levels in human A-549 cells following DENV4 infection. DENV4 infected A-549 cells were collected daily for 5 days, and RNA was extracted to quantify viral load. Gene expression of Drosha, Dicer, and DGCR8 was determined using quantitative PCR (RT-qPCR). We found that DENV4 infection exhibited the highest viral load 3 days post-infection. Dicer, Drosha, and DGCR8 showed reduced expression following DENV4 infection as compared with negative controls. In addition, we hypothesize that reduced expression of DGCR8 may not only be related to miRNA biogenesis, but also other small RNAs. This study may change our understanding regarding the relationship between host cells and the dengue virus.

  4. Temporal regulation of mRNAs for select bone morphogenetic proteins (BMP), BMP receptors and their associated SMAD proteins during bovine early embryonic development: effects of exogenous BMP2 on embryo developmental progression

    PubMed Central

    2014-01-01

    blastocyst mRNA for CDX2 and NANOG. Conclusions Abundance of maternally derived mRNAs for above BMP system components are dynamically regulated during oocyte maturation and early embryogenesis. Exogenous BMP2 treatment does not influence progression to various developmental endpoints, but impacts characteristics of resulting blastocysts. Results support a potential role for BMPs in bovine early embryogenesis. PMID:25027287

  5. An integrated approach identifies IFN-regulated microRNAs and targeted mRNAs modulated by different HCV replicon clones

    PubMed Central

    2011-01-01

    Background Infections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-β (IFN-β) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system). Methods The expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction. Results The expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection. Conclusions The present findings reveal that 3 IFN-β-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use

  6. Translation initiation of viral mRNAs.

    PubMed

    López-Lastra, Marcelo; Ramdohr, Pablo; Letelier, Alejandro; Vallejos, Maricarmen; Vera-Otarola, Jorge; Valiente-Echeverría, Fernando

    2010-05-01

    Viruses depend on cells for their replication but have evolved mechanisms to achieve this in an efficient and, in some instances, a cell-type-specific manner. The expression of viral proteins is frequently subject to translational control. The dominant target of such control is the initiation step of protein synthesis. Indeed, during the early stages of infection, viral mRNAs must compete with their host counterparts for the protein synthetic machinery, especially for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition viruses use diverse strategies so that ribosomes can be recruited selectively to viral mRNAs. In this review we focus on the initiation of protein synthesis and outline some of the strategies used by viruses to ensure efficient translation initiation of their mRNAs.

  7. A Novel Role of Vimentin Filaments: Binding and Stabilization of Collagen mRNAs

    PubMed Central

    Challa, Azariyas A.; Stefanovic, Branko

    2011-01-01

    The stem-loop in the 5′ untranslated region (UTR) of collagen α1(I) and α2(I) mRNAs (5′SL) is the key element regulating their stability and translation. Stabilization of collagen mRNAs is the predominant mechanism for high collagen expression in fibrosis. LARP6 binds the 5′SL of α1(I) and α2(I) mRNAs with high affinity. Here, we report that vimentin filaments associate with collagen mRNAs in a 5′SL- and LARP6-dependent manner and stabilize collagen mRNAs. LARP6 interacts with vimentin filaments through its La domain and colocalizes with the filaments in vivo. Knockdown of LARP6 by small interfering RNA (siRNA) or mutation of the 5′SL abrogates the interaction of collagen mRNAs with vimentin filaments. Vimentin knockout fibroblasts produce reduced amounts of type I collagen due to decreased stability of collagen α1(I) and α2(I) mRNAs. Disruption of vimentin filaments using a drug or by expression of dominant-negative desmin reduces type I collagen expression, primarily due to decreased stability of collagen mRNAs. RNA fluorescence in situ hybridization (FISH) experiments show that collagen α1(I) and α2(I) mRNAs are associated with vimentin filaments in vivo. Thus, vimentin filaments may play a role in the development of tissue fibrosis by stabilizing collagen mRNAs. This finding will serve as a rationale for targeting vimentin in the development of novel antifibrotic therapies. PMID:21746880

  8. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs.

    PubMed

    Giraldez, Antonio J; Mishima, Yuichiro; Rihel, Jason; Grocock, Russell J; Van Dongen, Stijn; Inoue, Kunio; Enright, Anton J; Schier, Alexander F

    2006-04-07

    MicroRNAs (miRNAs) comprise 1 to 3% of all vertebrate genes, but their in vivo functions and mechanisms of action remain largely unknown. Zebrafish miR-430 is expressed at the onset of zygotic transcription and regulates morphogenesis during early development. By using a microarray approach and in vivo target validation, we find that miR-430 directly regulates several hundred target messenger RNA molecules (mRNAs). Most targets are maternally expressed mRNAs that accumulate in the absence of miR-430. We also show that miR-430 accelerates the deadenylation of target mRNAs. These results suggest that miR-430 facilitates the deadenylation and clearance of maternal mRNAs during early embryogenesis.

  9. Functional signature for the recognition of specific target mRNAs by human Staufen1 protein

    PubMed Central

    de Lucas, Susana; Oliveros, Juan Carlos; Chagoyen, Mónica; Ortín, Juan

    2014-01-01

    Cellular messenger RNAs (mRNAs) are associated to proteins in the form of ribonucleoprotein particles. The double-stranded RNA-binding (DRB) proteins play important roles in mRNA synthesis, modification, activity and decay. Staufen is a DRB protein involved in the localized translation of specific mRNAs during Drosophila early development. The human Staufen1 (hStau1) forms RNA granules that contain translation regulation proteins as well as cytoskeleton and motor proteins to allow the movement of the granule on microtubules, but the mechanisms of hStau1-RNA recognition are still unclear. Here we used a combination of affinity chromatography, RNAse-protection, deep-sequencing and bioinformatic analyses to identify mRNAs differentially associated to hStau1 or a mutant protein unable to bind RNA and, in this way, defined a collection of mRNAs specifically associated to wt hStau1. A common sequence signature consisting of two opposite-polarity Alu motifs was present in the hStau1-associated mRNAs and was shown to be sufficient for binding to hStau1 and hStau1-dependent stimulation of protein expression. Our results unravel how hStau1 identifies a wide spectrum of cellular target mRNAs to control their localization, expression and fate. PMID:24470147

  10. Different mRNAs have different nuclear transit times in Dictyostelium discoideum aggregates.

    PubMed Central

    Mangiarotti, G; Zuker, C; Chisholm, R L; Lodish, H F

    1983-01-01

    Nuclear processing of mRNA precursors in differentiating multicellular Dictyostelium discoideum aggregates is markedly slower than in growing amoebae. Thus, we have been able to determine the time of nuclear processing of individual mRNA species in postaggregating cells by following the incorporation of 32PO4 into nuclear and cytoplasmic RNA complementary to cloned cDNAs. Precursors of mRNAs synthesized during both growth and differentiation remain in the nucleus for about 25 to 60 min. By contrast, typical mRNAs which are synthesized only by postaggregative cells have nuclear processing times between 50 and 100 min. Depending on the particular mRNA, between 20 and 60% of nuclear transcripts are converted into cytoplasmic mRNA. A third class of mRNAs are transcribed from a set of repetitive DNA segments and are expressed predominantly during differentiation. Nuclear precursors of these mRNAs are extensively degraded within the nucleus or very rapidly after transport to the cytoplasm. Those sequences that are stable in the cytoplasm exit from the nucleus only after a lag of over 2 h. Thus, mRNAs encoded by different genes that are subject to different types of developmental controls display different times of transit to the cytoplasm and different efficiencies of nuclear processing. Differential nuclear processing may contribute to the regulation of the level of individual cytoplasmic mRNAs. Images PMID:6621537

  11. Translational coregulation of 5'TOP mRNAs by TIA-1 and TIAR.

    PubMed

    Damgaard, Christian Kroun; Lykke-Andersen, Jens

    2011-10-01

    The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5'-terminal oligopyrimidine tracts (5'TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5'TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5'TOP mRNA regulation, which upon amino acid starvation assemble onto the 5' end of 5'TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5'TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5'TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment.

  12. Emerging Roles and Landscape of Translating mRNAs in Plants.

    PubMed

    Sablok, Gaurav; Powell, Jonathan J; Kazan, Kemal

    2017-01-01

    Plants use a wide range of mechanisms to adapt to different environmental stresses. One of the earliest responses displayed under stress is rapid alterations in stress responsive gene expression that has been extensively analyzed through expression profiling such as microarrays and RNA-sequencing. Recently, expression profiling has been complemented with proteome analyses to establish a link between transcriptional and the corresponding translational changes. However, proteome profiling approaches have their own technical limitations. More recently, ribosome-associated mRNA profiling has emerged as an alternative and a robust way of identifying translating mRNAs, which are a set of mRNAs associated with ribosomes and more likely to contribute to proteome abundance. In this article, we briefly review recent studies that examined the processes affecting the abundance of translating mRNAs, their regulation during plant development and tolerance to stress conditions and plant factors affecting the selection of translating mRNA pools. This review also highlights recent findings revealing differential roles of alternatively spliced mRNAs and their translational control during stress adaptation. Overall, better understanding of processes involved in the regulation of translating mRNAs has obvious implications for improvement of stress tolerance in plants.

  13. Emerging Roles and Landscape of Translating mRNAs in Plants

    PubMed Central

    Sablok, Gaurav; Powell, Jonathan J.; Kazan, Kemal

    2017-01-01

    Plants use a wide range of mechanisms to adapt to different environmental stresses. One of the earliest responses displayed under stress is rapid alterations in stress responsive gene expression that has been extensively analyzed through expression profiling such as microarrays and RNA-sequencing. Recently, expression profiling has been complemented with proteome analyses to establish a link between transcriptional and the corresponding translational changes. However, proteome profiling approaches have their own technical limitations. More recently, ribosome-associated mRNA profiling has emerged as an alternative and a robust way of identifying translating mRNAs, which are a set of mRNAs associated with ribosomes and more likely to contribute to proteome abundance. In this article, we briefly review recent studies that examined the processes affecting the abundance of translating mRNAs, their regulation during plant development and tolerance to stress conditions and plant factors affecting the selection of translating mRNA pools. This review also highlights recent findings revealing differential roles of alternatively spliced mRNAs and their translational control during stress adaptation. Overall, better understanding of processes involved in the regulation of translating mRNAs has obvious implications for improvement of stress tolerance in plants. PMID:28919899

  14. Evaluating the Stability of mRNAs and Noncoding RNAs.

    PubMed

    Ayupe, Ana Carolina; Reis, Eduardo M

    2017-01-01

    Changes in RNA stability have an important impact in the gene expression regulation. Different methods based on the transcription blockage with RNA polymerase inhibitors or metabolic labeling of newly synthesized RNAs have been developed to evaluate RNA decay rates in cultured cell. Combined with techniques to measure transcript abundance genome-wide, these methods have been used to reveal novel features of the eukaryotic transcriptome. The stability of protein-coding mRNAs is in general closely associated to the physiological function of their encoded proteins, with short-lived mRNAs being significantly enriched among regulatory genes whereas genes associated with housekeeping functions are predominantly stable. Likewise, the stability of noncoding RNAs (ncRNAs) seems to reflect their functional role in the cell. Thus, investigating RNA stability can provide insights regarding the function of yet uncharacterized regulatory ncRNAs. In this chapter, we discuss the methodologies currently used to estimate RNA decay and outline an experimental protocol for genome-wide estimation of RNA stability of protein-coding and lncRNAs. This protocol details the transcriptional blockage of cultured cells with actinomycin D, followed by RNA isolation at different time points, the determination of transcript abundance by qPCR/DNA oligoarray hybridization, and the calculation of individual transcript half-lives.

  15. IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

    PubMed

    Zhou, Hao; Bulek, Katarzyna; Li, Xiao; Herjan, Tomasz; Yu, Minjia; Qian, Wen; Wang, Han; Zhou, Gao; Chen, Xing; Yang, Hui; Hong, Lingzi; Zhao, Junjie; Qin, Luke; Fukuda, Koichi; Flotho, Annette; Gao, Ji; Dongre, Ashok; Carman, Julie A; Kang, Zizhen; Su, Bing; Kern, Timothy S; Smith, Jonathan D; Hamilton, Thomas A; Melchior, Frauke; Fox, Paul L; Li, Xiaoxia

    2017-10-09

    Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1 binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

  16. Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana.

    PubMed

    Juntawong, Piyada; Sorenson, Reed; Bailey-Serres, Julia

    2013-06-01

    RNA binding proteins (RBPs) function post-transcriptionally to fine-tune gene regulation. Arabidopsis thaliana has four Gly-rich, zinc finger-containing RBPs called cold shock proteins 1-4 (CSP1-CSP4), that possess an evolutionary conserved cold shock domain. Here, we determined that CSP1 associates with polyribosomes (polysomes) via an RNA-mediated interaction. Both the abundance and polysomal co-fractionation of CSP1 was enhanced in the cold (4°C), but did not influence global levels of polysomes, which were minimally perturbed by above freezing cold temperatures. Using a polyclonal antiserum, CSP1 was co-immunopurified with several hundred transcripts from rosettes of plants cultivated at 23°C or transferred to 4°C for 12 h. CSP1-associated mRNAs were characterized by G+C-rich 5' untranslated regions and gene ontologies related to cellular respiration, mRNA binding and translation. The majority of the CSP1-associated mRNAs were constitutively expressed and stable in the cold. CSP1 abundance was correlated with improved translation of ribosomal protein mRNAs during cold stress and improved maintenance of homeostasis and translation of mRNAs under water-deficit stress. In summary, CSP1 selectively chaperones mRNAs, providing translational enhancement during stress.

  17. Systematic imaging reveals features and changing localization of mRNAs in Drosophila development

    PubMed Central

    Jambor, Helena; Surendranath, Vineeth; Kalinka, Alex T; Mejstrik, Pavel; Saalfeld, Stephan; Tomancak, Pavel

    2015-01-01

    mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types—the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3′UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells. DOI: http://dx.doi.org/10.7554/eLife.05003.001 PMID:25838129

  18. Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.

    PubMed

    Jambor, Helena; Surendranath, Vineeth; Kalinka, Alex T; Mejstrik, Pavel; Saalfeld, Stephan; Tomancak, Pavel

    2015-04-02

    mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

  19. Regulation of synaptic Pumilio function by an aggregation-prone domain

    PubMed Central

    Salazar, Anna M.; Silverman, Edward J.; Menon, Kaushiki P.; Zinn, Kai

    2010-01-01

    We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities might be regulated by assembly into ordered aggregates. The search algorithm was based on evolutionary sequence conservation patterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N-terminus, and the complete PUF-9 protein localize to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin+ trait, which is transmitted by a heritable aggregate. NQ1 also assembles into amyloid fibrils in vitro. In Drosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality and NMJ phenotypes caused by overexpression of Pum in muscles. The Q/N block of NQ1 is required for these phenotypic effects. Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a “dominant-negative” effect, in which it would block Q/N-mediated interactions of Pum with itself or with cofactors required for translational repression. PMID:20071514

  20. N6-methyladenosine is required for the hypoxic stabilization of specific mRNAs

    PubMed Central

    Fry, Nate J.; Law, Brittany A.; Ilkayeva, Olga R.

    2017-01-01

    Post-transcriptional regulation of mRNA during oxygen deprivation, or hypoxia, can affect the survivability of cells. Hypoxia has been shown to increase stability of a subset of ischemia-related mRNAs, including VEGF. RNA binding proteins and miRNAs have been identified as important for post-transcriptional regulation of individual mRNAs, but corresponding mechanisms that regulate global stability are not well understood. Recently, mRNA modification by N6-methyladenosine (m6A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability and promotion of translation, but the role of m6A in the hypoxia response is unknown. In this study, we investigate the effect of hypoxia on RNA modifications including m6A. Our results show hypoxia increases m6A content of poly(A)+ messenger RNA (mRNA), but not in total or ribosomal RNA in HEK293T cells. Using m6A mRNA immunoprecipitation, we identify specific hypoxia-modified mRNAs, including glucose transporter 1 (Glut1) and c-Myc, which show increased m6A levels under hypoxic conditions. Many of these mRNAs also exhibit increased stability, which was blocked by knockdown of m6A-specific methyltransferases METTL3/14. However, the increase in mRNA stability did not correlate with a change in translational efficiency or the steady-state amount of their proteins. Knockdown of METTL3/14 did reveal that m6A is involved in recovery of translational efficiency after hypoxic stress. Therefore, our results suggest that an increase in m6A mRNA during hypoxic exposure leads to post-transcriptional stabilization of specific mRNAs and contributes to the recovery of translational efficiency after hypoxic stress. PMID:28611253

  1. Multiple Export Mechanisms for mRNAs

    PubMed Central

    Delaleau, Mildred; Borden, Katherine L. B.

    2015-01-01

    Nuclear mRNA export plays an important role in gene expression. We describe the mechanisms of mRNA export including the importance of mRNP assembly, docking with the nuclear basket of the nuclear pore complex (NPC), transit through the central channel of the NPC and cytoplasmic release. We describe multiple mechanisms of mRNA export including NXF1 and CRM1 mediated pathways. Selective groups of mRNAs can be preferentially transported in order to respond to cellular stimuli. RNAs can be selected based on the presence of specific cis-acting RNA elements and binding of specific adaptor proteins. The role that dysregulation of this process plays in human disease is also discussed. PMID:26343730

  2. The race to decipher the top secrets of TOP mRNAs.

    PubMed

    Meyuhas, Oded; Kahan, Tamar

    2015-07-01

    Cells encountering hostile growth conditions, like those residing in the middle of a newly developing solid tumor, conserve resources and energy by downregulating protein synthesis. One mechanism in this response is the translational repression of multiple mRNAs that encode components of the translational apparatus. This coordinated translational control is carried through a common cis-regulatory element, the 5' Terminal OligoPyrimidine motif (5'TOP), after which these mRNAs are referred to as TOP mRNAs. Subsequent to the initial structural and functional characterization of members of this family, the research of TOP mRNAs has progressed in three major directions: a) delineating the landscape of the family; b) establishing the pathways that transduce stress cues into selective translational repression; and c) attempting to decipher the most proximal trans-acting factor(s) and defining its mode of action--a repressor or activator. The present chapter critically reviews the development in these three avenues of research with a special emphasis on the two "top secrets" of the TOP mRNA family: the scope of its members and the identity of the proximal cellular regulator(s). This article is part of a Special Issue entitled: Translation and Cancer.

  3. Reassessment of the Role of TSC, mTORC1 and MicroRNAs in Amino Acids-Meditated Translational Control of TOP mRNAs

    PubMed Central

    Patursky-Polischuk, Ilona; Kasir, Judith; Miloslavski, Rachel; Hayouka, Zvi; Hausner-Hanochi, Mirit; Stolovich-Rain, Miri; Tsukerman, Pinchas; Biton, Moshe; Mudhasani, Rajini; Jones, Stephen N.; Meyuhas, Oded

    2014-01-01

    TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation. PMID:25338081

  4. Reassessment of the role of TSC, mTORC1 and microRNAs in amino acids-meditated translational control of TOP mRNAs.

    PubMed

    Patursky-Polischuk, Ilona; Kasir, Judith; Miloslavski, Rachel; Hayouka, Zvi; Hausner-Hanochi, Mirit; Stolovich-Rain, Miri; Tsukerman, Pinchas; Biton, Moshe; Mudhasani, Rajini; Jones, Stephen N; Meyuhas, Oded

    2014-01-01

    TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

  5. Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

    PubMed Central

    Damgaard, Christian Kroun; Lykke-Andersen, Jens

    2011-01-01

    The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5′-terminal oligopyrimidine tracts (5′TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5′TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5′TOP mRNA regulation, which upon amino acid starvation assemble onto the 5′ end of 5′TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5′TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5′TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment. PMID:21979918

  6. Vertebrate mRNAs with a 5'-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element.

    PubMed Central

    Avni, D; Shama, S; Loreni, F; Meyuhas, O

    1994-01-01

    The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing cells. This response is mediated through a regulatory element residing in the 5' untranslated region of these mRNAs and includes a 5' terminal oligopyrimidine tract (5' TOP). To further characterize the translational cis-regulatory element, we monitored the translational behavior of various endogenous and heterologous mRNAs or hybrid transcripts derived from transfected chimeric genes. The translational efficiency of these mRNAs was assessed in cells that either were growing normally or were growth arrested under various physiological conditions. Our experiments have yielded the following results: (i) the translation of mammalian rp mRNAs is properly regulated in amphibian cells, and likewise, amphibian rp mRNA is regulated in mammalian cells, indicating that all of the elements required for translation control of rp mRNAs are conserved among vertebrate classes; (ii) selective translational control is not confined to rp mRNAs, as mRNAs encoding the naturally occurring ubiquitin-rp fusion protein and elongation factor 1 alpha, which contain a 5' TOP, also conform this mode of regulation; (iii) rat rpP2 mRNA contains only five pyrimidines in its 5' TOP, yet this mRNA is translationally controlled in the same fashion as other rp mRNAs with a 5' TOP of eight or more pyrimidines; (iv) full manifestation of this mode of regulation seems to require both the 5' TOP and sequences immediately downstream; and (v) an intact translational regulatory element from rpL32 mRNA fails to exert its regulatory properties even when preceded by a single A residue. Images PMID:8196625

  7. Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

    PubMed Central

    Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P.; Romby, Pascale; Springer, Mathias; Marzi, Stefano

    2013-01-01

    Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

  8. nanoCAGE reveals 5′ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs

    PubMed Central

    Gandin, Valentina; Masvidal, Laia; Hulea, Laura; Gravel, Simon-Pierre; Cargnello, Marie; McLaughlan, Shannon; Cai, Yutian; Balanathan, Preetika; Morita, Masahiro; Rajakumar, Arjuna; Furic, Luc; Pollak, Michael; Porco, John A.; St-Pierre, Julie; Pelletier, Jerry; Larsson, Ola; Topisirovic, Ivan

    2016-01-01

    The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5′ TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5′ TOP motif but that 5′ UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5′ UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5′ UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5′ UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5′ UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5′ UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells. PMID:26984228

  9. nanoCAGE reveals 5' UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs.

    PubMed

    Gandin, Valentina; Masvidal, Laia; Hulea, Laura; Gravel, Simon-Pierre; Cargnello, Marie; McLaughlan, Shannon; Cai, Yutian; Balanathan, Preetika; Morita, Masahiro; Rajakumar, Arjuna; Furic, Luc; Pollak, Michael; Porco, John A; St-Pierre, Julie; Pelletier, Jerry; Larsson, Ola; Topisirovic, Ivan

    2016-05-01

    The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5' TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5' TOP motif but that 5' UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5' UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5' UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5' UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5' UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5' UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells. © 2016 Gandin et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

    PubMed Central

    Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

    2015-01-01

    Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. PMID:25941326

  11. Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice.

    PubMed

    Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

    2015-07-01

    Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings.

  12. UTRdb: a specialized database of 5'- and 3'-untranslated regions of eukaryotic mRNAs.

    PubMed Central

    Pesole, G; Liuni, S; Grillo, G; Saccone, C

    1998-01-01

    The important role the untranslated regions of eukaryotic mRNAs may play in gene regulation and expression is now widely acknowledged. For this reason we developed UTRdb, a specialized database of 5'- and 3'-untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases, including the presence of functional patterns already demonstrated by experimental analysis to have some functional role. A collection of such patterns is being collected in UTRsite database (http://bio-www.ba.cnr.it:8000/srs5/) which can also be used with appropriate computational tools to detect known functional patterns contained in mRNA untranslated regions. PMID:9399833

  13. UTRdb: a specialized database of 5' and 3' untranslated regions of eukaryotic mRNAs.

    PubMed Central

    Pesole, G; Liuni, S; Grillo, G; Ippedico, M; Larizza, A; Makalowski, W; Saccone, C

    1999-01-01

    The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb (http://bigarea.area.ba.cnr.it:8000/BioWWW/#U TRdb), a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. PMID:9847176

  14. High throughput sequencing analysis of Trypanosoma brucei DRBD3/PTB1-bound mRNAs.

    PubMed

    Das, Anish; Bellofatto, Vivian; Rosenfeld, Jeffrey; Carrington, Mark; Romero-Zaliz, Rocío; del Val, Coral; Estévez, Antonio M

    2015-01-01

    Trypanosomes are early-branched eukaryotes that show an unusual dependence on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins are crucial in controlling mRNA fate in these organisms, but their RNA substrates remain largely unknown. Here we have analyzed on a global scale the mRNAs associated with the Trypanosoma brucei RNA-binding protein DRBD3/PTB1, by capturing ribonucleoprotein complexes using UV cross-linking and subsequent immunoprecipitation. DRBD3/PTB1 associates with many transcripts encoding ribosomal proteins and translation factors. Consequently, silencing of DRBD3/PTB1 expression altered the protein synthesis rate. DRBD3/PTB1 also binds to mRNAs encoding the enzymes required to obtain energy through the oxidation of proline to succinate. We hypothesize that DRBD3/PTB1 is a key player in RNA regulon-based gene control influencing protein synthesis in trypanosomes.

  15. Expression profile of long noncoding RNAs and mRNAs in peripheral blood mononuclear cells from myasthenia gravis patients.

    PubMed

    Zhang, Fang; Liu, Guiyou; Bu, Yali; Ma, Xiaofeng; Hao, Junwei

    2016-10-15

    For the epigenetic characterization of myasthenia gravis (MG), we determined whether long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) are expressed differentially in subjects with and without MG. Compared with healthy control subjects, the MG patients had 1561 upregulated lncRNAs, 1034 downregulated lncRNAs, 921 upregulated mRNAs, and 806 downregulated mRNAs (fold change>2.0). Several GO terms including nucleic acid transcription factor activity, inflammatory response, regulation of leukocyte activation, lymphocyte proliferation and regulation of B cell proliferation were enriched in gene lists, suggesting a potential correlation with MG. Pathway analysis then demonstrated that cytokine-cytokine receptor interaction, intestinal immune network for lgA production, NOD-like receptor signaling pathway, NF-kappaB signaling pathway, cell adhesion molecules and TNF signaling pathway play important roles in MG. Co-expression network analysis indicated that 33 lncRNAs were predicted to have 31 cis-regulated target genes, and 65 lncRNAs appeared to regulate the patients' 45 trans target genes among differentially expressed lncRNAs. Our present study identified a subset of dysregulated lncRNAs and mRNAs in patients with MG, which may impact this disease process. Copyright © 2016. Published by Elsevier B.V.

  16. Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite.

    PubMed

    Belusko, P B; Nakajima, T; Azuma, M; Shearer, T R

    2003-10-13

    An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.

  17. Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula.

    PubMed

    Reynoso, Mauricio Alberto; Blanco, Flavio Antonio; Bailey-Serres, Julia; Crespi, Martín; Zanetti, María Eugenia

    2013-01-01

    Translation of mRNAs is a key regulatory step that contributes to the coordination and modulation of eukaryotic gene expression during development or adaptation to the environment. mRNA stability or translatability can be regulated by the action of small regulatory RNAs (sRNAs), which control diverse biological processes. Under low nitrogen conditions, leguminous plants associate with soil bacteria and develop a new organ specialized in nitrogen fixation: the nodule. To gain insight into the translational regulation of mRNAs during nodule formation, the association of mRNAs and sRNAs to polysomes was characterized in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Quantitative comparison of steady-state and polysomal mRNAs for 15 genes involved in nodulation identified a group of transcripts with slight or no change in total cellular abundance that were significantly upregulated at the level of association with polysomes in response to rhizobia. This group included mRNAs encoding receptors like kinases required either for nodule organogenesis, bacterial infection or both, and transcripts encoding GRAS and NF-Y transcription factors (TFs). Quantitative analysis of sRNAs in total and polysomal RNA samples revealed that mature microRNAs (miRNAs) were associated with the translational machinery, notably, miR169 and miR172, which target the NF-YA/HAP2 and AP2 TFs, respectively. Upon inoculation, levels of miR169 pronouncedly decreased in polysomal complexes, concomitant with the increased accumulation of the NF-YA/HAP2 protein. These results indicate that both mRNAs and miRNAs are subject to differential recruitment to polysomes, and expose the importance of selective mRNA translation during root nodule symbiosis.

  18. Spatial and temporal translational control of germ cell mRNAs mediated by the eIF4E isoform IFE-1.

    PubMed

    Friday, Andrew J; Henderson, Melissa A; Morrison, J Kaitlin; Hoffman, Jenna L; Keiper, Brett D

    2015-12-15

    Regulated mRNA translation is vital for germ cells to produce new proteins in the spatial and temporal patterns that drive gamete development. Translational control involves the de-repression of stored mRNAs and their recruitment by eukaryotic initiation factors (eIFs) to ribosomes. C. elegans expresses five eIF4Es (IFE-1-IFE-5); several have been shown to selectively recruit unique pools of mRNA. Individual IFE knockouts yield unique phenotypes due to inefficient translation of certain mRNAs. Here, we identified mRNAs preferentially translated through the germline-specific eIF4E isoform IFE-1. Differential polysome microarray analysis identified 77 mRNAs recruited by IFE-1. Among the IFE-1-dependent mRNAs are several required for late germ cell differentiation and maturation. Polysome association of gld-1, vab-1, vpr-1, rab-7 and rnp-3 mRNAs relies on IFE-1. Live animal imaging showed IFE-1-dependent selectivity in spatial and temporal translation of germline mRNAs. Altered MAPK activation in oocytes suggests dual roles for IFE-1, both promoting and suppressing oocyte maturation at different stages. This single eIF4E isoform exerts positive, selective translational control during germ cell differentiation.

  19. Molecular expression of aquaporin mRNAs in the northern house mosquito, Culex pipiens.

    PubMed

    Yang, Liu; Piermarini, Peter M

    2017-01-01

    Aquaporins (AQPs) are a family of membrane-bound proteins, originally described as water-channels, that broadly exist in prokaryotic and eukaryotic cells. However, some AQPs can transport small molecules (e.g. urea, glycerol) along with or preferentially to water. Previous work in the mosquitoes Aedes aegypti and Anopheles gambiae have characterized the molecular expression of one or more AQP genes and shown that they are involved in water homeostasis after blood feeding, tolerance to dehydration and heat stresses, and development of Plasmodium falciparum in the mosquito. However, the molecular expression of AQPs in the northern house mosquito Culex pipiens, the primary vector of West Nile virus, is presently unknown. Here we characterize the relative mRNA expression of six AQP genes in C. pipiens. We used quantitative RT-PCR (qPCR) to compare AQP mRNA levels 1) among different life stages and sexes, 2) between blood fed and non-blood fed adult females, 3) between adult females injected with phosphate-buffered saline (PBS) and non-injected females, and 4) between adult females exposed to a low vs. high relative humidity. We found that: 1) four AQP mRNAs (Drip, Bib, Eglp1, Aqp12L) were differentially expressed between larval and adult life stages, but not between sexes; 2) after a blood meal, the expression of two AQP mRNAs (Drip and Aqp12L) were down-regulated and one AQP mRNA (Eglp2) was up-regulated; 3) the expression of two AQP mRNAs (Drip and Prip) were down-regulated and one AQP mRNA (Eglp1) was up regulated after injecting the hemolymph of mosquitoes with PBS; and 4) the expression of Prip and Eglp2 mRNAs were both down-regulated in response to a low relative humidity. Our study is the first to characterize the molecular expression patterns of AQPs in C. pipiens, thus providing a foundation for future research elucidating the physiological and functional roles of AQPs in this important disease vector. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

    PubMed

    Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

    2014-06-04

    Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

  1. Differential dendritic targeting of AMPA receptor subunit mRNAs in adult rat hippocampal principal neurons and interneurons.

    PubMed

    Cox, David J; Racca, Claudia

    2013-06-15

    In hippocampal neurons, AMPA receptors (AMPARs) mediate fast excitatory postsynaptic responses at glutamatergic synapses, and are involved in various forms of synaptic plasticity. Dendritic local protein synthesis of selected AMPAR subunit mRNAs is considered an additional mechanism to independently and rapidly control the strength of individual synapses. We have used fluorescent in situ hybridization and immunocytochemistry to analyze the localization of AMPAR subunit (GluA1-4) mRNAs and their relationship with the translation machinery in principal cells and interneurons of the adult rat hippocampus. The mRNAs encoding all four AMPAR subunits were detected in the somata and dendrites of CA3 and CA1 pyramidal cells and those of six classes of CA1 γ-aminobutyric acid (GABA)ergic interneurons. GluA1-4 subunit mRNAs were highly localized to the apical dendrites of pyramidal cells, whereas in interneurons they were present in multiple dendrites. In contrast, in the dentate gyrus, GluA1-4 subunit mRNAs were virtually restricted to the somata and were absent from the dendrites of granule cells. These different regional and cell type-specific labeling patterns also correlated with the localization of markers for components of the protein synthesis machinery. Our results support the local translation of GluA1-4 mRNAs in dendrites of hippocampal pyramidal cells and CA1 interneurons but not in granule cells of the dentate gyrus. Furthermore, the regional and cell type-specific differences we observed suggest that each cell type uses distinct ways of regulating the local translation of AMPAR subunits.

  2. Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/- mice.

    PubMed

    Morita, Masahiro; Oike, Yuichi; Nagashima, Takeshi; Kadomatsu, Tsuyoshi; Tabata, Mitsuhisa; Suzuki, Toru; Nakamura, Takahisa; Yoshida, Nobuaki; Okada, Mariko; Yamamoto, Tadashi

    2011-09-06

    Obesity is a life-threatening factor and is often associated with dysregulation of gene expression. Here, we show that the CNOT3 subunit of the CCR4-NOT deadenylase complex is critical to metabolic regulation. Cnot3(+/-) mice are lean with hepatic and adipose tissues containing reduced levels of lipids, and show increased metabolic rates and enhanced glucose tolerance. Cnot3(+/-) mice remain lean and sensitive to insulin even on a high-fat diet. Furthermore, introduction of Cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs is not altered in Cnot3(+/-) vis-à-vis wild-type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism-related PDK4 and IGFBP1, are increased in Cnot3(+/-) hepatocytes, having poly(A) tails that are longer than those seen in control cells. We provide evidence that CNOT3 is involved in recruitment of the CCR4-NOT deadenylase to the 3' end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decrease upon fasting, we propose that CNOT3 responds to feeding conditions to regulate deadenylation-specific mRNAs and energy metabolism.

  3. Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/− mice

    PubMed Central

    Morita, Masahiro; Oike, Yuichi; Nagashima, Takeshi; Kadomatsu, Tsuyoshi; Tabata, Mitsuhisa; Suzuki, Toru; Nakamura, Takahisa; Yoshida, Nobuaki; Okada, Mariko; Yamamoto, Tadashi

    2011-01-01

    Obesity is a life-threatening factor and is often associated with dysregulation of gene expression. Here, we show that the CNOT3 subunit of the CCR4–NOT deadenylase complex is critical to metabolic regulation. Cnot3+/− mice are lean with hepatic and adipose tissues containing reduced levels of lipids, and show increased metabolic rates and enhanced glucose tolerance. Cnot3+/− mice remain lean and sensitive to insulin even on a high-fat diet. Furthermore, introduction of Cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs is not altered in Cnot3+/− vis-à-vis wild-type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism-related PDK4 and IGFBP1, are increased in Cnot3+/− hepatocytes, having poly(A) tails that are longer than those seen in control cells. We provide evidence that CNOT3 is involved in recruitment of the CCR4–NOT deadenylase to the 3′ end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decrease upon fasting, we propose that CNOT3 responds to feeding conditions to regulate deadenylation-specific mRNAs and energy metabolism. PMID:21897366

  4. FRET-Based Identification of mRNAs Undergoing Translation

    PubMed Central

    Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L.; Smilansky, Zeev; Cooperman, Barry S.; Goldman, Yale E.

    2012-01-01

    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed. PMID:22693619

  5. FRET-based identification of mRNAs undergoing translation.

    PubMed

    Stevens, Benjamin; Chen, Chunlai; Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L; Smilansky, Zeev; Cooperman, Barry S; Goldman, Yale E

    2012-01-01

    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

  6. Hypermethylated-capped selenoprotein mRNAs in mammals

    PubMed Central

    Wurth, Laurence; Gribling-Burrer, Anne-Sophie; Verheggen, Céline; Leichter, Michael; Takeuchi, Akiko; Baudrey, Stéphanie; Martin, Franck; Krol, Alain; Bertrand, Edouard; Allmang, Christine

    2014-01-01

    Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5′-end m7G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5′-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo. PMID:25013170

  7. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    PubMed Central

    THOMSEN, RUNE; LIBRI, DOMENICO; BOULAY, JOCELYNE; ROSBASH, MICHAEL; JENSEN, TORBEN HEICK

    2003-01-01

    In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal site of transcription, and known S. cerevisiae nuclear structures such as the nucleolus and the nucleolar body. Our results show that retained SSA4 RNA localizes to an area in close proximity to the SSA4 locus. On deletion of Rrp6p and release from the genomic locus, heat shock mRNAs produced in the rat7–1 strain colocalize predominantly with nucleolar antigens. Bulk poly(A)+ RNA, on the other hand, is localized primarily to the nuclear rim. Interestingly, the RNA binding nucleocytoplasmic shuttle protein Npl3p shows strong colocalization with bulk poly(A)+ RNA, regardless of its nuclear location. Taken together, our data show that retention occurs close to the gene and indicate distinct nuclear fates of different mRNAs. PMID:12923254

  8. LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs

    PubMed Central

    Ruzzenente, Benedetta; Metodiev, Metodi D; Wredenberg, Anna; Bratic, Ana; Park, Chan Bae; Cámara, Yolanda; Milenkovic, Dusanka; Zickermann, Volker; Wibom, Rolf; Hultenby, Kjell; Erdjument-Bromage, Hediye; Tempst, Paul; Brandt, Ulrich; Stewart, James B; Gustafsson, Claes M; Larsson, Nils-Göran

    2012-01-01

    Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine-rich pentatricopeptide repeat containing (LRPPRC) protein regulates mitochondrial mRNA stability and an amino-acid substitution of this protein causes the French-Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue-specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady-state levels of most mitochondrial mRNAs. LRPPRC forms an RNA-dependent protein complex that is necessary for maintaining a pool of non-translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post-transcriptional level. PMID:22045337

  9. Differential compartmentalization of mRNAs in squid giant axon.

    PubMed

    Chun, J T; Gioio, A E; Crispino, M; Giuditta, A; Kaplan, B B

    1996-11-01

    Previously, we reported that the squid giant axon contains a heterogeneous population of mRNAs that includes beta-actin, beta-tubulin, kinesin, neurofilament proteins, and enolase. To define the absolute levels and relative distribution of these mRNAs, we have used competitive reverse transcription-PCR to quantify the levels of five mRNAs present in the giant axon and giant fiber lobe (GFL), the location of the parental cell soma. In the GFL, the number of transcripts for these mRNAs varied over a fourfold range, with beta-tubulin being the most abundant mRNA species (1.25 x 10(9) molecules per GFL). Based on transcript number, the rank order of mRNA levels in the GFL was beta-tubulin > beta-actin > kinesin > enolase > microtubule-associated protein (MAP) H1. In contrast, kinesin mRNA was most abundant in the axon (4.1 x 10(7) molecules per axon) with individual mRNA levels varying 15-fold. The rank order of mRNA levels in the axon was kinesin > beta-tubulin > MAP H1 > beta-actin > enolase. The relative abundance of the mRNA species in the axon did not correlate with the size of the transcript, nor was it directly related to their corresponding levels in the GFL. Taken together, these findings confirm that significant amounts of mRNA are present in the giant axon and suggest that specific mRNAs are differentially transported into the axonal domain.

  10. Distinct expression of Cbln family mRNAs in developing and adult mouse brains.

    PubMed

    Miura, Eriko; Iijima, Takatoshi; Yuzaki, Michisuke; Watanabe, Masahiko

    2006-08-01

    Cbln1 belongs to the C1q and tumour necrosis factor superfamily, and plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although Cbln2-Cbln4 are also expressed in the brain and could form heteromeric complexes with Cbln1, their precise expressions remain unclear. Here, we investigated gene expression of the Cbln family in developing and adult C57BL mouse brains by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and high-resolution in situ hybridization (ISH) analyses. In the adult brain, spatial patterns of mRNA expression were highly differential depending on Cbln subtypes. Notably, particularly high levels of Cbln mRNAs were expressed in some nuclei and neurons, whereas their postsynaptic targets often lacked or were low for any Cbln mRNAs, as seen for cerebellar granule cells/Purkinje cells, entorhinal cortex/hippocampus, intralaminar group of thalamic nuclei/caudate-putamen, and dorsal nucleus of the lateral lemniscus/central nucleus of the inferior colliculus. In the developing brain, Cbln1, 2, and 4 mRNAs appeared as early as embryonic day 10-13, and exhibited transient up-regulation during the late embryonic and neonatal periods. For example, Cbln2 mRNA was expressed in the cortical plate of the developing neocortex, displaying a high rostromedial to low caudolateral gradient. In contrast, Cbln3 mRNA was selective to cerebellar granule cells throughout development, and its onset was as late as postnatal day 7-10. These results will provide a molecular-anatomical basis for future studies that characterize roles played by the Cbln family.

  11. Gene-expression reversal of lncRNAs and associated mRNAs expression in active vs latent HIV infection

    PubMed Central

    Nair, Madhavan; Sagar, Vidya; Pilakka-Kanthikeel, Sudheesh

    2016-01-01

    Interplay between lncRNAs and mRNAs is rapidly emerging as a key epigenetic mechanism in controlling various cell functions. HIV can actively infect and/or can persist latently for years by manipulating host epigenetics; however, its molecular essence remains undiscovered in entirety. Here for the first time, we delineate the influence of HIV on global lncRNAs expression in monocytic cells lines. Our analysis revealed the expression modulation of nearly 1060 such lncRNAs which are associated with differentially expressed mRNAs in active and latent infection. This suggests a greater role of lncRNAs in regulating transcriptional and post-transcriptional gene expression during HIV infection. The differentially expressed mRNAs were involved in several different biological pathways where immunological networks were most enriched. Importantly, we discovered that HIV induces expression reversal of more than 150 lncRNAs between its active and latent infection. Also, hundreds of unique lncRNAs were identified in both infection conditions. The pathology specific “gene-expression reversal” and “on-and-off” switching of lncRNAs and associated mRNAs may lead to establish the relationship between active and HIV infection. PMID:27756902

  12. Mature mRNAs accumulated in the nucleus are neither the molecules in transit to the cytoplasm nor constitute a stockpile for gene expression.

    PubMed Central

    Weil, D; Boutain, S; Audibert, A; Dautry, F

    2000-01-01

    In higher eukaryotes, the regulation of pre-mRNA processing is still poorly known. The accumulation of various mature mRNAs, which can be observed in the nuclei of mammalian cells, is suggestive of a regulatory role of transport. However, the significance of these nuclear mRNA is presently unknown. We have used a tetracycline-regulated promoter to investigate the dynamics of these pools of mRNAs upon arrest of transcription. We observed, for beta-globin and LT-alpha genes, a slow disappearance of these mRNA from the nucleus, with an apparent half-life that is similar to their cytoplasmic half-life. In view of these dynamics, these mRNA cannot simply be mature mRNAs in transit to the cytoplasm. They could be mRNAs retained in the nucleus, provided that the regulation of mRNA stability is comparable in the nucleus and the cytoplasm. But, because of their limited stability, these nuclear mRNAs cannot constitute a significant stock for gene expression. Alternatively, they could reflect a bidirectional transport of mRNA, that is, to and from the cytoplasm, which would provide a direct explanation for the similarity in both compartments of their half-life and poly(A) tail shortening over time. PMID:10917593

  13. Protein synthesis inhibitors enhance the expression of mRNAs for early inducible inflammatory genes via mRNA stabilization.

    PubMed

    Yamazaki, Soh; Takeshige, Koichiro

    2008-02-01

    Expression of inflammatory genes is regulated at multiple steps, including transcriptional activation and mRNA stabilization. During an investigation into the requirement of de novo protein synthesis for the induction of inflammatory genes, it was revealed that protein synthesis inhibitors unexpectedly potentiated the induction of mRNAs for primary response genes, while the inhibitors suppressed the induction of secondary inducible genes as previously described. Stimulus-induced nuclear translocation and promoter recruitment of NF-kappaB, which is responsible for the transcriptional activation of many inflammatory genes, were largely unaffected by the inhibitors. Instead, these inhibitors prolonged the half-lives of all of the primary inducible mRNAs tested. Thus, these findings emphasize the important contribution of regulated mRNA longevity to gene expression induced by pro-inflammatory stimulation.

  14. Transcriptome-Wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and Noncoding RNAs.

    PubMed

    David, Rakesh; Burgess, Alice; Parker, Brian; Li, Jun; Pulsford, Kalinya; Sibbritt, Tennille; Preiss, Thomas; Searle, Iain Robert

    2017-03-01

    Posttranscriptional methylation of RNA cytosine residues to 5-methylcytosine (m(5)C) is an important modification with diverse roles, such as regulating stress responses, stem cell proliferation, and RNA metabolism. Here, we used RNA bisulfite sequencing for transcriptome-wide quantitative mapping of m(5)C in the model plant Arabidopsis thaliana We discovered more than a thousand m(5)C sites in Arabidopsis mRNAs, long noncoding RNAs, and other noncoding RNAs across three tissue types (siliques, seedling shoots, and roots) and validated a number of these sites. Quantitative differences in methylated sites between these three tissues suggest tissue-specific regulation of m(5)C. Perturbing the RNA m(5)C methyltransferase TRM4B resulted in the loss of m(5)C sites on mRNAs and noncoding RNAs and reduced the stability of tRNA(Asp(GTC)) We also demonstrate the importance of m(5)C in plant development, as trm4b mutants have shorter primary roots than the wild type due to reduced cell division in the root apical meristem. In addition, trm4b mutants show increased sensitivity to oxidative stress. Finally, we provide insights into the targeting mechanism of TRM4B by demonstrating that a 50-nucleotide sequence flanking m(5)C C3349 in MAIGO5 mRNA is sufficient to confer methylation of a transgene reporter in Nicotiana benthamiana.

  15. Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide.

    PubMed

    Shi, Zhen; Fujii, Kotaro; Kovary, Kyle M; Genuth, Naomi R; Röst, Hannes L; Teruel, Mary N; Barna, Maria

    2017-07-06

    Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae

    PubMed Central

    Walters, Robert W.; Matheny, Tyler; Mizoue, Laura S.; Rao, Bhalchandra S.; Muhlrad, Denise; Parker, Roy

    2017-01-01

    RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5′ nicotinamide-adenine dinucleotide (NAD+) RNA in bacteria. Whether 5′ NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5′ NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae. NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5′ end processed. These results define an additional 5′ RNA cap structure in eukaryotes and raise the possibility that this 5′ NAD+ cap could modulate RNA stability and translation on specific subclasses of mRNAs. PMID:28031484

  17. Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs.

    PubMed

    Zinoviev, Alexandra; Hellen, Christopher U T; Pestova, Tatyana V

    2015-03-19

    Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated "termination upstream ribosomal binding site" (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2(∗). To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs

    PubMed Central

    Zinoviev, Alexandra; Hellen, Christopher U. T.; Pestova, Tatyana V.

    2015-01-01

    Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long ORFs on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG designated “termination upstream ribosomal binding site” (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2*. To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes, and diminishes dependence on eIF3 of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes. PMID:25794616

  19. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  20. A four-nucleotide translation enhancer in the 3'-terminal consensus sequence of the nonpolyadenylated mRNAs of rotavirus.

    PubMed Central

    Chizhikov, V; Patton, J T

    2000-01-01

    The 5' cap and poly(A) tail of eukaryotic mRNAs work synergistically to enhance translation through a process that requires interaction of the cap-associated eukaryotic initiation factor, eIF-4G, and the poly(A)-binding protein, PABP. Because the mRNAs of rotavirus, and other members of the Reoviridae, contain caps but lack poly(A) tails, their translation may be enhanced through a unique mechanism. To identify translation-enhancement elements in the viral mRNAs that stimulate translation in vivo, chimeric RNAs were prepared that contained an open reading frame for luciferase and the 5' and 3' untranslated regions (UTRs) of a rotavirus mRNA or of a nonviral mRNA. Transfection of the chimeric RNAs into rotavirus-infected cells showed that the viral 3' UTR contained a translation-enhancement element that promoted gene expression. The element did not enhance gene expression in uninfected cells and did not affect the stability of the RNAs. Mutagenesis showed that the conserved sequence GACC located at the 3' end of rotavirus mRNAs operated as an enhancement element. The 3'-GACC element stimulated protein expression independently of the sequence of the 5' UTR, although efficient expression required the RNA to contain a cap. The results indicate that the expression of viral proteins in rotavirus-infected cells is specifically up-regulated by the activity of a novel 4-nt 3' translation enhancer (TE) common to the 11 nonpolyadenylated mRNAs of the virus. The 4-nt sequence of the rotavirus 3' TE represents by far the shortest of any of the sequence enhancers known to stimulate translation. PMID:10864041

  1. Patterns of gene expression in the murine brain revealed by in situ hybridization of brain-specific mRNAs.

    PubMed

    Branks, P L; Wilson, M C

    1986-07-01

    Biochemical differences between neuronal cell populations of the mammalian brain, including selection of neurotransmitters and distinct neural antigens, suggest that the regulation of gene expression plays an important role in defining brain function. Here we describe the use of in situ hybridization to identify cDNA clones of highly regulated mRNA species and to define directly their pattern of gene expression in brain at both gross morphological and cellular levels. One of the selected cDNA clones, pMuBr2, detected a single 3.0 kb mRNA species, which from in situ hybridization appears specific to oligodendroglia cells. Three other cDNA clones, pMuBr3, 8 and 85, identified polyadenylated mRNA transcripts expressed by neuronal cells of the murine brain. Viewed at the gross morphological level, the mRNAs hybridizing to these cDNA sequences exhibit different patterns of abundance distinguishing such brain structures as pons, anterior thalamus, hippocampus, basal ganglia and anterior lobe of the neuroendocrine pituitary gland. At the cellular level, in situ hybridization revealed that these mRNAs are differentially expressed by morphologically and functionally distinct neurons of the cerebellum and hippocampal formation. When examined in the context of known brain function, however, the regulated expression of the neuron-specific mRNAs does not correlate simply with known cellular morphology or previously demonstrated neuronal relationships suggesting novel patterns of gene expression which may contribute to brain function.

  2. Quail Pax-6 (Pax-QNR) mRNAs are expressed from two promoters used differentially during retina development and neuronal differentiation.

    PubMed Central

    Plaza, S; Dozier, C; Turque, N; Saule, S

    1995-01-01

    During investigations on the regulation of the Pax-6 gene, we characterized a cDNA from quail neuroretina showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, we localized this second quail Pax-6 promoter, termed P1. As reported for the already described P0 promoter, P1 was also transactivated in vitro by the p46Pax-QNR protein. RNase protection assays performed with quail neuroretina RNA showed that P1-initiated mRNAs were detected before the P0-initiated mRNAs, remained constant up to embryonic day 8, and decreased slowly thereafter whereas, P0-initiated mRNAs accumulated up to embryonic day 8. In contrast, quail retinal pigmented epithelium expressed only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induced neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, expressed Pax-QNR from the P0 promoter. These results suggest that expression of the quail Pax-6 gene is under the control of different regulators through alternate promoters, P0 being activated at the onset of neuronal differentiation. PMID:7760830

  3. Somatomedin-C/insulin-like growth factor-I and Insulin-like growth factor-II mRNAs in rate fetal and adult tissues

    SciTech Connect

    Lund, P.K.; Moats-Staats, B.M.; Hynes, M.A.; Simmons, J.G.; Jansen, M.; D'ercole, A.J.; Van Wyk, J.J.

    1986-11-05

    Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study /sup 32/P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyze rat Sm-C/IGF-I and IGF-II mRNAs in poly(A/sup +/) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobase (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A/sup +/) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded.

  4. Comprehensive analysis of lncRNAs and mRNAs in skeletal muscle of rainbow trout (Oncorhynchus mykiss) exposed to estradiol

    USDA-ARS?s Scientific Manuscript database

    Estradiol (E2) is a steroid hormone that negatively affects muscle growth in rainbow trout, but the mechanisms directing with this response are not fully understood. To better characterize the effects of E2 in muscle, we identified differentially regulated mRNAs and lncRNAs in juvenile rainbow trout...

  5. Comprehensive analysis of lncRNAs and mRNAs in skeletal muscle of rainbow trout (Oncorhynchus mykiss) exposed to estradiol.

    PubMed

    Wang, Jian; Koganti, Prasanthi P; Yao, Jianbo; Wei, Shuo; Cleveland, Beth

    2017-09-18

    Estradiol (E2) is a steroid hormone that negatively affects muscle growth in rainbow trout (Oncorhynchus mykiss), but the mechanisms directing with this response are not fully understood. To better characterize the effects of E2 in muscle, we identified differentially regulated mRNAs and lncRNAs in juvenile rainbow trout exposed to E2. Here, we performed next-generation RNA sequencing and comprehensive bioinformatics analyses to characterize the transcriptome profiles, including mRNAs and long noncoding RNAs (lncRNAs), in skeletal muscle of rainbow trout injected with E2. A total of 226 lncRNAs and 253 mRNAs were identified as differentially regulated. We identified crucial pathways, including several signal transduction pathways, hormone response, oxidative response and protein, carbon and fatty acid metabolism pathways. Subsequently, a functional lncRNA-mRNA co-expression network was constructed, which consisted of 681 co-expression relationships between 164 lncRNAs and 201 mRNAs. Moreover, a lncRNA-pathway network was constructed. A total of 65 key lncRNAs were identified that regulate 20 significantly enriched pathways. Overall, our analysis provides insights into mRNA and lncRNA networks in rainbow trout skeletal muscle and their regulation by E2 while understanding the molecular mechanism of lncRNAs.

  6. LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs

    PubMed Central

    Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

    2017-01-01

    The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5’TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5’TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5’ and 3’UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5’UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3’UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.25237.001 PMID:28650797

  7. Autoregulated changes in stability of polyribosome-bound. beta. -tubulin mRNAs are specified by the first 13 translated nucleotides

    SciTech Connect

    Yen, T.J.; Gay, D.A.; Pachter, J.S.; Cleveland, D.W.

    1988-03-01

    The expression of tubulin polypeptides in animal cells is controlled by an autoregulatory mechanism whereby increases in the tubulin subunit concentration result in rapid and specific degradation of tubulin mRNAs. The authors have now determined that the sequences that are necessary and sufficient to specify mouse ..beta..-tubulin mRNAs as substrates for this autoregulated instability reside within the first 13 translated nucleotides (which encode the first four ..beta..-tubulin amino acids Met-Arg-Glu-Ile). This domain has been functionally conserved throughout evolution, inasmuch as sequences isolated from the analogous region of human, chicken, and yeast ..beta..-tubulin mRNAs also confer autoregulation. Further, for an RNA to be a substrate for regulation, not only must it carry the 13-nucleotide coding sequence, but it must also be ribosome bound and its translation must proceed 3' to codon 41.

  8. Splicing and Polyadenylation of Human Papillomavirus Type 16 mRNAs

    PubMed Central

    Wu, Chengjun; Kajitani, Naoko; Schwartz, Stefan

    2017-01-01

    The human papillomavirus type 16 (HPV16) life cycle can be divided into an early stage in which the HPV16 genomic DNA is replicated, and a late stage in which the HPV16 structural proteins are synthesized and virions are produced. A strong coupling between the viral life cycle and the differentiation state of the infected cell is highly characteristic of all HPVs. The switch from the HPV16 early gene expression program to the late requires a promoter switch, a polyadenylation signal switch and a shift in alternative splicing. A number of cis-acting RNA elements on the HPV16 mRNAs and cellular and viral factors interacting with these elements are involved in the control of HPV16 gene expression. This review summarizes our knowledge of HPV16 cis-acting RNA elements and cellular and viral trans-acting factors that regulate HPV16 gene expression at the level of splicing and polyadenylation. PMID:28208770

  9. Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters.

    PubMed

    Chen, Hao; Zuo, Qisheng; Wang, Yingjie; Song, Jiuzhou; Yang, Huilin; Zhang, Yani; Li, Bichun

    2017-02-13

    Traditional approaches for generating goat pluripotent stem cells (iPSCs) suffer from complexity and low preparation efficiency. Therefore, we tried to derive goat iPSCs with a new method by transfecting exogenous Oct4, Sox2, Klf4 and c-Myc mRNAs into goat embryonic fibroblasts (GEFs), and explore the mechanisms regarding the transcription regulation of the reprogramming factors in goat iPSCs induction. mRNAs of the four reprogramming factors were transfected into GEFs, and were localized in nucleus with approximately 90% transfection efficiency. After five consecutive transfections, GEFs tended to aggregate by day 10. Clones appeared on day 15-18, and typical embryonic stem cell -like clones formed on day 20. One thousand AKP staining positive clones were achieved in 10(4) GEFs, with approximately 1.0% induction efficiency. Immunofluorescence staining and qRT-PCR detection of the ESCs markers confirmed the properties of the goat iPSCs. The achieved goat iPSCs could be cultured to 22nd passage, which showed normal karyotype. The goat iPSCs were able to differentiate into embryoid bodies with three germ layers. qRT-PCR and western blot showed activated endogenous pluripotent factors expression in the later phase of mRNA-induced goat iPSCs induction. Epigenetic analysis of the endogenous pluripotent gene Nanog revealed its demethylation status in derived goat iPSCs. Core promoter regions of the four reprogramming factors were determined. Transcription factor binding sites, including Elf-1, AP-2, SP1, C/EBP and MZF1, were identified to be functional in the core promoter regions of these reprogramming genes. Demethylation and deacetylation of the promoters enhanced their transcription activities. We successfully generated goat iPSCs by transfection of Oct4, Sox2, Klf4 and c-Myc mRNAs into GEFs, which initiated the endogenous reprogramming network and altered the methylation status of pluripotent genes. Core promoter regions and functional transcription binding sites of

  10. Glucocorticoids and the expression of mRNAs for neurotrophins, their receptors and GAP-43 in the rat hippocampus.

    PubMed

    Chao, H M; McEwen, B S

    1994-10-01

    The genes encoding brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and basic fibroblast growth factor (bFGF) are all expressed in the adult rat hippocampus. The colocalization of the these factors with the receptors to which they bind, namely trkB, trkC and the bFGF receptor, respectively, suggests that in the hippocampus they may exert their putative protective and trophic effects through an autocrine mechanism. The morphology and survival of hippocampal neurons are also affected by glucocorticoids, which can act as transcriptional activators of gene expression. In this study we have used in situ hybridization to investigate the adrenal steroid regulation of the mRNAs encoding the neurotrophic factors BDNF, NT-3, and bFGF, their respective receptors, and the growth-associated protein GAP-43. After 7 days of adrenalectomy (ADX), there was an increase in the level of GAP-43 mRNA expression in the CA1 and CA3 pyramidal cell layers of the hippocampus, that was prevented by corticosterone replacement to the ADX animals. In the CA2 subregion, adrenalectomy resulted in a decrease in bFGF mRNA expression, that was reversed by steroid treatment. There was evidence for glucocorticoid modulation of the BDNF and NT-3 mRNAs in pyramidal cell layers and in the dentate gyrus, but not of the mRNAs encoding the trkB, trk C or bFGF receptors.

  11. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed Central

    Mangiarotti, Giorgio

    2003-01-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs. PMID:12392449

  12. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed

    Mangiarotti, Giorgio

    2003-03-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs.

  13. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons.

    PubMed

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2008-11-10

    Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.

  14. Dynamic co-expression network analysis of lncRNAs and mRNAs associated with venous congestion

    PubMed Central

    Li, Jinshun; Xu, Yuqin; Xu, Jia; Wang, Jinhua; Wu, Liying

    2016-01-01

    Venous congestion and volume overload are important in cardiorenal syndromes, in which multiple regulated factors are involved, including long non-coding RNAs (lncRNAs). To investigate the underlying role of lncRNAs in regulating the development of venous congestion, an Affymetrix microarray associated with peripheral venous congestion was annotated, then a bipartite dynamic lncRNA-mRNA co-expression network was constructed in which nodes indicated lncRNAs or mRNAs. The nodes were connected when the lncRNAs or mRNAs were dynamically co-expressed. Following functional analysis of this network, several dynamic alternative pathways were identified, including the calcium signaling pathway during venous congestion development. Additionally, certain lncRNAs (LINC00523, LINC01210 and RP11-435O5.5) were identified that may potentially dynamically regulate certain proteins, including plasma membrane calcium ATPase (PMCA) and G protein-coupled receptor (GPCR), in the calcium signaling pathway. Particularly, the dynamically regulated switch of LINC00523 from co-expression with PMCA to GPCR may be involved in damage to steady state intracellular calcium. In brief, the current study demonstrated a potential novel mechanism of lncRNA function during venous congestion. PMID:27431002

  15. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. 5’-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation

    PubMed Central

    Beck, Heather J.; Fleming, Ian M. C.

    2016-01-01

    Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs) that contain a conventional untranslated leader and Shine-Dalgarno (SD) sequence upstream of the gene’s start codon while also containing an AUG triplet at the mRNA’s 5’- terminus (5’-uAUG). Fusion of the coding sequence specified by the 5’-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5’-terminal upstream open reading frames (5’-uORFs) tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5’-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5’-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5’-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5’-uORFs may play roles in downstream regulation. Since the 5’-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5’-uAUGs should be examined for their potential to function also as leaderless mRNAs. PMID:27467758

  17. Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins

    PubMed Central

    Cridge, Andrew G.; Castelli, Lydia M.; Smirnova, Julia B.; Selley, Julian N.; Rowe, William; Hubbard, Simon J.; McCarthy, John E.G.; Ashe, Mark P.; Grant, Christopher M.; Pavitt, Graham D.

    2010-01-01

    eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3′-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought. PMID:20705650

  18. The CCR4-NOT Complex Mediates Deadenylation and Degradation of Stem Cell mRNAs and Promotes Planarian Stem Cell Differentiation

    PubMed Central

    Solana, Jordi; Gamberi, Chiara; Mihaylova, Yuliana; Grosswendt, Stefanie; Chen, Chen; Lasko, Paul; Rajewsky, Nikolaus; Aboobaker, A. Aziz

    2013-01-01

    Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology. PMID:24367277

  19. The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

    PubMed

    Solana, Jordi; Gamberi, Chiara; Mihaylova, Yuliana; Grosswendt, Stefanie; Chen, Chen; Lasko, Paul; Rajewsky, Nikolaus; Aboobaker, A Aziz

    2013-01-01

    Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

  20. Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes

    PubMed Central

    Jung, Volker; Beitzinger, Michaela; Nolte, Elke; Wach, Sven; Hart, Martin; Sapich, Sandra; Wiesehöfer, Marc; Wennemuth, Gunther; Eichner, Norbert; Stempfl, Thomas; Wullich, Bernd; Meister, Gunter

    2017-01-01

    Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs. PMID:28163933

  1. MicroRNAs Align With Accessible Sites in Target mRNAs

    PubMed Central

    Pan, Weihua; Xin, Ping; Clawson, Gary A.

    2015-01-01

    The importance of microRNAs (miRs) in control of gene expression is now clearly recognized. While individual microRNAs are thought to target hundreds of disparate mRNAs via imperfect base pairing, little is known about the characteristics of miR target sites. Here we show that the miRs can be aligned with empirically identified accessible sites in a target RNA (Cytokeratin 19, KRT), and that some of the aligned miRs functionally down-regulate KRT expression post-transcriptionally. We employed an RNase-H-based random library selection protocol to identify accessible sites in KRT RNA. We then aligned the Sanger Institute database collection of human miRs to KRT mRNA, and also aligned them using the web-based MicroInspector program. Most miRs aligned with the accessible sites identified empirically; those not aligned with the empirically identified sites also functioned effectively in RNase-H-based assays. Similar results were obtained with a second target RNA (Mammoglobin). Transient transfection assays established that some of the miRs which aligned with KRT significantly down-regulated it at the protein level, with no effect on RNA level. The functionally effective miRs aligned within the coding region of KRT, whereas a number of miRs which aligned with the 3′-untranslated region did not produce down-regulation. PMID:19998415

  2. RiboTag Analysis of Actively Translated mRNAs in Sertoli and Leydig Cells In Vivo

    PubMed Central

    Sanz, Elisenda; Evanoff, Ryan; Quintana, Albert; Evans, Elizabeth; Miller, Jeremy A.; Ko, Chemyong; Amieux, Paul S.; Griswold, Michael D.; McKnight, G. Stanley

    2013-01-01

    Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the “translatome” (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell. PMID:23776628

  3. Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction.

    PubMed

    McDermott, Suzanne M; Yang, Lu; Halstead, James M; Hamilton, Russell S; Meignin, Carine; Davis, Ilan

    2014-10-01

    Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.

  4. [Similar features in mechanisms of translation initiation of mRNAs in eukaryotic and prokaryotic systems].

    PubMed

    Andreev, D E; Terenin, I M; Dmitriev, S E; Shatskiĭ, I N

    2006-01-01

    Using as examples non-canonical features of translation initiation for some bacterial and mammalian mRNAs with unusual 5'- untranslated regions (5'-UTR) or lacking these regions (leaderless mRNAs), the authors of this review discuss similarities in mechanisms of translation initiation on prokaryotic and eukaryotic ribosomes.

  5. Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells.

    PubMed

    Yin, Wanzhong; Wang, Ping; Wang, Xin; Song, Wenzhi; Cui, Xiangyan; Yu, Hong; Zhu, Wei

    2013-06-01

    Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (≈ 45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33 ± 1.76 vs 28.75 ± 1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98 ± 0.52 vs 69.14 ± 0.89, P<0.05), increased efflux of rhodamine 123 (95.97 ± 0.56 vs 12.40 ± 0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

  6. Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

    PubMed Central

    Yin, Wanzhong; Wang, Ping; Wang, Xin; Song, Wenzhi; Cui, Xiangyan; Yu, Hong; Zhu, Wei

    2013-01-01

    Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer. PMID:23780424

  7. Detection of signals in mRNAs that influence translation.

    PubMed

    Brown, Chris M; Jacobs, Grant; Stockwell, Peter; Schreiber, Mark

    2003-01-01

    Genome sequencing efforts mean that we now have extensive data from a wide range of organisms to study. Understanding the differing natures of the biology of these organisms is an important aim of genome analysis. We are interested in signals that affect translation of mRNAs. Some signals in the mRNA influence how efficiently it is translated into protein. Previous studies have indicated that many important signals are located around the initiation and termination codons. We have developed tools described here to extract the relevant sequence regions from GenBank. To create databases organised by species, or higher taxonomic groupings (eg planta), a program was developed to dynamically view and edit the taxonomy database. Data from relevant species were then extracted using our Genbank feature table parser. We analysed all available sequences, particularly those from complete genomes. Patterns were then identified using information theory. The software is available from http://transterm.otago.ac.nz. Patterns around the initiation codons for most of the organisms fall into two groups, containing the previously known Shine-Dalgarno and Kozaks efficiency signals. However, we have identified several organisms that appear to utilise novel systems. Our analysis indicates that some organisms with extremely high GC% genomes do not have a strong dependence on base pairing ribosome binding sites, as the complementary sequence is absent from many genes.

  8. Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs.

    PubMed

    Abdelmohsen, Kotb; Tominaga, Kumiko; Lee, Eun Kyung; Srikantan, Subramanya; Kang, Min-Ju; Kim, Mihee M; Selimyan, Roza; Martindale, Jennifer L; Yang, Xiaoling; Carrier, France; Zhan, Ming; Becker, Kevin G; Gorospe, Myriam

    2011-10-01

    RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation. The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection. However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very rich in G residues and was not only found in the 3'-untranslated region (UTR), but also in the coding region (CR) and 5'-UTR. Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins. In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and enhances their translation.

  9. The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism

    PubMed Central

    Das, Anish; Morales, Rachel; Banday, Mahrukh; Garcia, Stacey; Hao, Li; Cross, George A.M.; Estevez, Antonio M.; Bellofatto, Vivian

    2012-01-01

    RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein–RNA interactions in vivo using UV light. Specific RBP42–mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism. PMID:22966087

  10. Sequestration of mRNAs Modulates the Timing of Translation during Meiosis in Budding Yeast.

    PubMed

    Jin, Liang; Zhang, Kai; Xu, Yifeng; Sternglanz, Rolf; Neiman, Aaron M

    2015-10-01

    Starvation of diploid cells of the budding yeast Saccharomyces cerevisiae induces them to enter meiosis and differentiate into haploid spores. During meiosis, the precise timing of gene expression is controlled at the level of transcription, and also translation. If cells are returned to rich medium after they have committed to meiosis, the transcript levels of most meiotically upregulated genes decrease rapidly. However, for a subset of transcripts whose translation is delayed until the end of meiosis II, termed protected transcripts, the transcript levels remain stable even after nutrients are reintroduced. The Ime2-Rim4 regulatory circuit controls both the delayed translation and the stability of protected transcripts. These protected mRNAs localize in discrete foci, which are not seen for transcripts of genes with different translational timing and are regulated by Ime2. These results suggest that Ime2 and Rim4 broadly regulate translational delay but that additional factors, such as mRNA localization, modulate this delay to tune the timing of gene expression to developmental transitions during sporulation.

  11. Sequestration of mRNAs Modulates the Timing of Translation during Meiosis in Budding Yeast

    PubMed Central

    Jin, Liang; Zhang, Kai; Xu, Yifeng; Sternglanz, Rolf

    2015-01-01

    Starvation of diploid cells of the budding yeast Saccharomyces cerevisiae induces them to enter meiosis and differentiate into haploid spores. During meiosis, the precise timing of gene expression is controlled at the level of transcription, and also translation. If cells are returned to rich medium after they have committed to meiosis, the transcript levels of most meiotically upregulated genes decrease rapidly. However, for a subset of transcripts whose translation is delayed until the end of meiosis II, termed protected transcripts, the transcript levels remain stable even after nutrients are reintroduced. The Ime2-Rim4 regulatory circuit controls both the delayed translation and the stability of protected transcripts. These protected mRNAs localize in discrete foci, which are not seen for transcripts of genes with different translational timing and are regulated by Ime2. These results suggest that Ime2 and Rim4 broadly regulate translational delay but that additional factors, such as mRNA localization, modulate this delay to tune the timing of gene expression to developmental transitions during sporulation. PMID:26217015

  12. Interplay of viral miRNAs and host mRNAs and proteins

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-10-01

    Recent experiments indicate that several viruses may encode microRNAs (miRNAs) in cells. Such RNAs may interfere with the host mRNAs and proteins. We present a kinetic analysis of this interplay. In our treatment, the viral miRNA is considered to be able to associate with the host mRNA with subsequent degradation. This process may result in a decline of the mRNA population and also in a decline of the population of the protein encoded by this mRNA. With these ingredients, we first show the types of the corresponding steady-state kinetics in the cases of positive and negative regulation of the miRNA synthesis by the protein. In addition, we scrutinize the situation when the protein regulates the virion replication or, in other words, provides a feedback for the replication. For the negative feedback, the replication rate is found to increase with increasing the intracellular virion population. For the positive feedback, the replication rate first increases and then drops. These features may determine the stability of steady states.

  13. Mitochondrial toxins in models of neurodegenerative diseases. II: Elevated zif268 transcription and independent temporal regulation of striatal D1 and D2 receptor mRNAs and D1 and D2 receptor-binding sites in C57BL/6 mice during MPTP treatment.

    PubMed

    Smith, T S; Trimmer, P A; Khan, S M; Tinklepaugh, D L; Bennett, J P

    1997-08-15

    Sporadic Parkinson's disease (PD) may arise from a defect in complex I of the mitochondrial electron transport chain (ETC), transmitted through mitochondrial DNA mutations. The N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of experimental PD is believed to arise from loss of complex I activity in dopamine (DA) neurons after accumulation of MPP+, a potent complex I inhibitor and the two electron monoamine oxidase B oxidation product of MPTP. Acute MPP+ infusion into striatum, possibly mimicking the in vivo situation after MPTP treatment, increases release of DA and production of hydroxyl radical (-OH). We treated C57BL/6 mice with MPTP and followed the expression of the immediate-early gene zif268 in striatum as a marker of DA synaptic activity, determined the pharmacology of its activation during MPTP toxicity, and assayed the time course of MPTP effects on striatal DA transporter (DAT), and D1 and D2 DA receptor-binding sites and their mRNAs. MPTP (24 mg/kg b.i.d. for 4 doses) increased striatal zif268 expression, with peak effects observed 24 h after starting MPTP. Increased striatal zif268 was dependent mainly on DA D1 and to a lesser extent on non-NMDA glutamate receptors and was not altered by inhibition of nitric oxide synthase (NOS). Our MPTP schedule resulted in a loss of about one-third of nigral DA neurons. We observed with [3H]mazindol autoradiography that loss of striatal DAT sites after starting MPTP was heterogenous and greatest in centromedial striatum, reached a maximum at 48 h and showed a slight recovery at 2 weeks. Striatal D1 and D2 receptor-binding sites (measured with [3H]SCH23390 and [3H]spiperone binding, respectively) and mRNA levels for D1 and D2 receptors (determined with quantitative in situ hybridization) were altered after MPTP treatment in temporally independent manners. MPTP toxicity to the nigrostriatal system likely induces substantial striatal DA release in vivo and stimulates transcription of at least one major IEG

  14. A cross-talk between Hepatitis B virus and host mRNAs confers viral adaptation to liver

    PubMed Central

    Hu, Jun; Xu, Yaxing; Li, Changfei; Hao, Junli; Peng, Shanxin; Chu, Xiaoyu; Zhang, Dake; Xu, Dongping; Meng, Songdong

    2015-01-01

    Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide. The replication of HBV which genome is only 3.2 kb long relies heavily on host factors. Previous studies demonstrated that a highly expressed liver-specific microRNA (miRNA) miR-122 suppresses HBV expression and replication in multiple ways. In this study, we found that the miR-122 response elements in viral genome facilitate HBV expression and replication in miR-122 highly-expressed hepatocytes. Moreover, mutations in miR-122 response elements are correlated with viral loads and disease progression in HBV-infected patients. We next found that HBV mRNA with miR-122 response elements alone could lead to altered expression of multiple host genes by whole genome expression analysis. HBV mRNA-mediated miR-122 down-regulation plays a major role in HBV mRNA-induced differential gene expression. HBV mRNA could enhance viral replication via miR-122 degradation and the up-regulation of its target cyclin G1. Our study thereby reveals that under the unique condition of high abundance of miR-122 and viral mRNAs and much lower level of miR-122 target in HBV infection, HBV may have evolved to employ the miRNA-mediated virus and host mRNAs network for optimal fitness within hepatocytes. PMID:26184825

  15. A catalog of stability-associated sequence elements in 3' UTRs of yeast mRNAs

    PubMed Central

    Shalgi, Reut; Lapidot, Michal; Shamir, Ron; Pilpel, Yitzhak

    2005-01-01

    Background In recent years, intensive computational efforts have been directed towards the discovery of promoter motifs that correlate with mRNA expression profiles. Nevertheless, it is still not always possible to predict steady-state mRNA expression levels based on promoter signals alone, suggesting that other factors may be involved. Other genic regions, in particular 3' UTRs, which are known to exert regulatory effects especially through controlling RNA stability and localization, were less comprehensively investigated, and deciphering regulatory motifs within them is thus crucial. Results By analyzing 3' UTR sequences and mRNA decay profiles of Saccharomyces cerevisiae genes, we derived a catalog of 53 sequence motifs that may be implicated in stabilization or destabilization of mRNAs. Some of the motifs correspond to known RNA-binding protein sites, and one of them may act in destabilization of ribosome biogenesis genes during stress response. In addition, we present for the first time a catalog of 23 motifs associated with subcellular localization. A significant proportion of the 3' UTR motifs is highly conserved in orthologous yeast genes, and some of the motifs are strikingly similar to recently published mammalian 3' UTR motifs. We classified all genes into those regulated only at transcription initiation level, only at degradation level, and those regulated by a combination of both. Interestingly, different biological functionalities and expression patterns correspond to such classification. Conclusion The present motif catalogs are a first step towards the understanding of the regulation of mRNA degradation and subcellular localization, two important processes which - together with transcription regulation - determine the cell transcriptome. PMID:16207357

  16. Diurnal Rhythmicity in the Pattern of mRNAs in the Leaves of Sinapis alba1

    PubMed Central

    Cremer, Frédéric; Dommes, Jacques; Van de Walle, Claude; Bernier, Georges

    1990-01-01

    Previous studies have shown that certain specific leaf mRNAs exhibit a diurnal rhythmicity in their quantity in higher plants. To determine whether this situation is restricted to a few mRNAs, or affects a large number, we have used in vitro translation and two-dimensional polyacrylamide gel electrophoresis to analyze the mRNA complement in leaves of Sinapis alba at different times during an 8-hour/16-hour day/night cycle. A method for the visual analysis of two-dimensional polyacrylamide gel electrophoresis was also developed. This method selected, at each sampling time, spots that were significant. It then selected, between two sampling times, intensity changes that were significant at the 0.02 confidence level. During a day/night cycle, complex rhythmic changes affected about 10% of the mRNAs. Nineteen different rhythm patterns were found. These 19 patterns fell into four main classes: mRNAs that increase during the light period and decrease during the dark, mRNAs that increase and then decrease during the light period, mRNAs that decrease during the light period and increase during the dark period, and mRNAs that increase and then decrease during the dark period. Images Figure 1 Figure 4 PMID:16667894

  17. RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei

    PubMed Central

    PELLETIER, MICHEL; READ, LAURIE K.

    2003-01-01

    RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by ∼98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%–80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs. PMID:12649497

  18. Transduction of Growth or Mitogenic Signals into Translational Activation of TOP mRNAs Is Fully Reliant on the Phosphatidylinositol 3-Kinase-Mediated Pathway but Requires neither S6K1 nor rpS6 Phosphorylation

    PubMed Central

    Stolovich, Miri; Tang, Hua; Hornstein, Eran; Levy, Galit; Cohen, Ruth; Bae, Sun Sik; Birnbaum, Morris J.; Meyuhas, Oded

    2002-01-01

    Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Bα (PKBα). Likewise, overexpression of constitutively active PI3-kinase or PKBα can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway. PMID:12417714

  19. Mapping circulating serum miRNAs to their immune-related target mRNAs

    PubMed Central

    Nosirov, Bakhtiyor; Billaud, Joël; Vandenbon, Alexis; Diez, Diego; Wijaya, Edward; Ishii, Ken J; Teraguchi, Shunsuke; Standley, Daron M

    2017-01-01

    Purpose Evidence suggests that circulating serum microRNAs (miRNAs) might preferentially target immune-related mRNAs. If this were the case, we hypothesized that immune-related mRNAs would have more predicted serum miRNA binding sites than other mRNAs and, reciprocally, that serum miRNAs would have more immune-related mRNA targets than non-serum miRNAs. Materials and methods We developed a consensus target predictor using the random forest framework and calculated the number of predicted miRNA–mRNA interactions in various subsets of miRNAs (serum, non-serum) and mRNAs (immune related, nonimmune related). Results Immune-related mRNAs were predicted to be targeted by serum miRNA more than other mRNAs. Moreover, serum miRNAs were predicted to target many more immune-related mRNA targets than non-serum miRNAs; however, these two biases in immune-related mRNAs and serum miRNAs appear to be completely independent. Conclusion Immune-related mRNAs have more miRNA binding sites in general, not just for serum miRNAs; likewise, serum miRNAs target many more mRNAs than non-serum miRNAs overall, regardless of whether they are immune related or not. Nevertheless, these two independent phenomena result in a significantly larger number of predicted serum miRNA–immune mRNA interactions than would be expected by chance. PMID:28203094

  20. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    PubMed

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

    2014-08-28

    Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

  1. Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells.

    PubMed

    Hong, Bok Sil; Cho, Ji-Hoon; Kim, Hyunjung; Choi, Eun-Jeong; Rho, Sangchul; Kim, Jongmin; Kim, Ji Hyun; Choi, Dong-Sic; Kim, Yoon-Keun; Hwang, Daehee; Gho, Yong Song

    2009-11-25

    Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation. These microvesicles can mediate communication between cells and affect various tumor-related processes in their target cells. We present potential roles of CRC cell-derived microvesicles in tumor progression via a global comparative microvesicular and cellular transcriptomic analysis of human SW480 CRC cells. We first identified 11,327 microvesicular mRNAs involved in tumorigenesis-related processes that reflect the physiology of donor CRC cells. We then found 241 mRNAs enriched in the microvesicles above donor cell levels, of which 27 were involved in cell cycle-related processes. Network analysis revealed that most of the cell cycle-related microvesicle-enriched mRNAs were associated with M-phase activities. The integration of two mRNA datasets showed that these M-phase-related mRNAs were differentially regulated across CRC patients, suggesting their potential roles in tumor progression. Finally, we experimentally verified the network-driven hypothesis by showing a significant increase in proliferation of endothelial cells treated with the microvesicles. Our study demonstrates that CRC cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells, suggesting that microvesicles of cancer cells can be involved in tumor growth and metastasis by facilitating angiogenesis-related processes. This information will help elucidate the pathophysiological functions of tumor-derived microvesicles, and aid in the development of cancer diagnostics, including colorectal cancer.

  2. A viral Sm-class RNA base-pairs with mRNAs and recruits microRNAs to inhibit apoptosis.

    PubMed

    Gorbea, Carlos; Mosbruger, Tim; Cazalla, Demián

    2017-10-04

    Viruses express several classes of non-coding RNAs; the functions and mechanisms by which most of these act are unknown. Herpesvirus saimiri, a γ-herpesvirus that establishes latency in the T cells of New World primates and has the ability to cause aggressive leukaemias and lymphomas in non-natural hosts, expresses seven small nuclear uracil-rich non-coding RNAs (called HSURs) in latently infected cells. These HSURs associate with Sm proteins, and share biogenesis and structural features with cellular Sm-class small nuclear RNAs. One of these HSURs (HSUR2) base-pairs with two host cellular microRNAs (miR-142-3p and miR-16) but does not affect their abundance or activity, which suggests that its interactions with them perform alternative functions. Here we show that HSUR2 also base-pairs with mRNAs in infected cells. We combined in vivo psoralen-mediated RNA-RNA crosslinking and high-throughput sequencing to identify the mRNAs targeted by HSUR2, which include mRNAs that encode retinoblastoma and factors involved in p53 signalling and apoptosis. We show that HSUR2 represses the expression of target mRNAs and that base-pairing between HSUR2 and miR-142-3p and miR-16 is essential for this repression, suggesting that HSUR2 recruits these two cellular microRNAs to its target mRNAs. Furthermore, we show that HSUR2 uses this mechanism to inhibit apoptosis. Our results uncover a role for this viral Sm-class RNA as a microRNA adaptor in the regulation of gene expression that follows precursor mRNA processing.

  3. Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart

    PubMed Central

    Pournajafi-Nazarloo, Hossein; Perry, Adam; Partoo, Leila; Papademeteriou, Eros; Azizi, Feridoun; Carter, C. Sue; Cushing, Bruce S.

    2007-01-01

    Oxytocin (OT) has been implicated in reproductive functions, induction of maternal behavior as well as endocrine and neuroendocrine regulation of the cardiovascular system. Here we demonstrate that neonatal manipulation of OT can modulate the mRNAs expression for OT receptor (OTR), atrial natriuretic peptide (ANP), endothelial nitric oxide synthase (eNOS) and estrogen receptor alpha (ERα) in the heart. On the first day of postnatal life, female and male rats were randomly assigned to receive one of following treatments; (a) 50 µl i.p. injection of 7 µg OT, (b) 0.7 µg of OT antagonist (OTA), or (c) isotonic saline (SAL). Hearts were collected either on postnatal day 1 or day 21 (D1 or D21) and the mRNAs expression of OTR, ANP, inducible NOS (iNOS), eNOS, ERα and estrogen receptor beta (ERβ) were compared by age, treatment, and sex utilizing Real Time PCR. OT treatment significantly increased heart OTR, ANP and eNOS mRNAs expression on D1 in both males and females, ERα increased only in females. While there were significant changes in the relative expression of all types of mRNA between D1 and D21 there were no significant treatment effects observed in D21 animals. OTA treatment significantly decreased basal ANP and eNOS mRNAs expression on D1 in both sexes. The results indicate that during the early postnatal period OT can have an immediate effect on the expression OTR, ANP, eNOS, and ERα mRNAs and that these effects are mitigated by D21. Also with the exception of ERα mRNA, the effects are the same in both sexes. PMID:17537544

  4. IMP-3 protects the mRNAs of cyclins D1 and D3 from GW182/AGO2-dependent translational repression.

    PubMed

    Deforzh, Evgeny; Vargas, Thaiz Rivera; Kropp, Jeremie; Vandamme, Marie; Pinna, Guillaume; Polesskaya, Anna

    2016-12-01

    IGF-2 mRNA binding protein 3 (IGF2BP3, IMP-3) is a well-known post-transcriptional regulatory factor of gene expression, mainly involved in embryonic development and oncogenesis. We have previously demonstrated that a subset of IMP-3 targets, such as the mRNAs of cyclins D1, D3 and G1, are positively regulated by IMP-3, and that this regulation depends on nuclear localization of IMP-3. In the present study, we show that as a first step following a knock-down of IMP-3, the protein levels of the cyclins rapidly decrease, while their mRNAs remain stable and associated with the polyribosomes, though not translated. We have elucidated the molecular mechanisms of this regulation, demonstrating that IMP-3 and its protein partners ILF3/NF90 and PTBP1 bind to the 3'UTRs of the cyclin mRNAs and protect them from the translational repression induced by miRNA-dependent recruitment of AGO2/GW182 complex in human cancer cells.

  5. Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes

    SciTech Connect

    Kartasova, T.; Ponec, M.; van de Putte, P.

    1988-02-01

    uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with (/sup 35/S)methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.

  6. Herpes simplex virus ICP27 increases translation of a subset of viral late mRNAs.

    PubMed

    Fontaine-Rodriguez, Errin C; Knipe, David M

    2008-04-01

    The herpes simplex virus (HSV) ICP27 immediate-early protein plays an essential role in the expression of viral late genes. ICP27 is a multifunctional protein and has been reported to regulate multiple steps of mRNA synthesis and processing, including transcription, splicing, and nuclear export. Recently, ICP27 was reported to interact with translation factors and to stimulate translation of the viral late mRNA encoding VP16. We examined the effects of ICP27 on accumulation, nuclear export, and translation of HSV 1 (HSV-1) late mRNAs encoding VP16, ICP5, and gD. We confirm here that ICP27 stimulates translation of VP16 mRNA as well as an additional HSV-1 late ICP5 mRNA. The data presented here demonstrate that translation levels of both VP16 and ICP5 mRNA is reduced during infections with the ICP27-null virus mutant d27-1, and with ICP27 C-terminal deletion mutant viruses n406 and n504, compared to wild-type virus. In contrast, the translation of gD mRNA is not affected by the presence of ICP27 during infection. These data demonstrate that ICP27 functions to increase the translation levels of a subset of HSV-1 late genes, and this function requires the C terminus of ICP27.

  7. Acetylcholine receptor alpha-subunit and myogenin mRNAs in thymus and thymomas.

    PubMed Central

    Kornstein, M. J.; Asher, O.; Fuchs, S.

    1995-01-01

    Myasthenia gravis is an autoimmune disorder characterized in most cases by serological antibody against the acetylcholine receptor (AChR). Evidence for intrathymic localization of AChR suggests that the thymus has an important role in the pathogenesis of this disorder. Using reverse transcription followed by the polymerase chain reaction, we have demonstrated AChR alpha-subunit mRNA in thymuses and thymomas from patients with and without myasthenia gravis. We have also studied the expression of myogenin which is known to be involved in the regulation of AChR expression. By using the reverse transcription polymerase chain reaction, we found myogenin mRNAs in all of the thymuses and thymomas. Thus, both AChR alpha-subunit and myogenin mRNA are present in all of these specimens. By immunohistochemistry myoid cells (desmin and myoglobin positive) were present in all (four of four) thymuses studied and in two of five thymomas. Thus, in thymomas, nonmyoid cells might express both AChR and myogenin. These results indicate that cells within the thymus and thymoma express AChR and its regulatory protein myogenin and that such cells, under certain conditions, might play a role in the triggering of myasthenia gravis. Images Figure 2 Figure 3 PMID:7778671

  8. Expression Variations of miRNAs and mRNAs in Rice (Oryza sativa)

    PubMed Central

    Wen, Ming; Xie, Munan; He, Lian; Wang, Yushuai; Shi, Suhua; Tang, Tian

    2016-01-01

    Differences in expression levels are an important source of phenotypic variation within and between populations. MicroRNAs (miRNAs) are key players in post-transcriptional gene regulation that are important for plant development and stress responses. We surveyed expression variation of miRNAs and mRNAs of six accessions from two rice subspecies Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica using deep sequencing. While more than half (53.7%) of the mature miRNAs exhibit differential expression between grains and seedlings of rice, only 11.0% show expression differences between subspecies, with an additional 2.2% differentiated for the development-by-subspecies interaction. Expression variation is greater for lowly conserved miRNAs than highly conserved miRNAs, whereas the latter show stronger negative correlation with their targets in expression changes between subspecies. Using a permutation test, we identified 51 miRNA–mRNA pairs that correlate negatively or positively in expression level among cultivated rice. Genes involved in various metabolic processes and stress responses are enriched in the differentially expressed genes between rice indica and japonica subspecies. Our results indicate that stabilizing selection is the major force governing miRNA expression in cultivated rice, albeit positive selection may be responsible for much of the between-subspecies expression divergence. PMID:27797952

  9. Expression Variations of miRNAs and mRNAs in Rice (Oryza sativa).

    PubMed

    Wen, Ming; Xie, Munan; He, Lian; Wang, Yushuai; Shi, Suhua; Tang, Tian

    2016-12-31

    Differences in expression levels are an important source of phenotypic variation within and between populations. MicroRNAs (miRNAs) are key players in post-transcriptional gene regulation that are important for plant development and stress responses. We surveyed expression variation of miRNAs and mRNAs of six accessions from two rice subspecies Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica using deep sequencing. While more than half (53.7%) of the mature miRNAs exhibit differential expression between grains and seedlings of rice, only 11.0% show expression differences between subspecies, with an additional 2.2% differentiated for the development-by-subspecies interaction. Expression variation is greater for lowly conserved miRNAs than highly conserved miRNAs, whereas the latter show stronger negative correlation with their targets in expression changes between subspecies. Using a permutation test, we identified 51 miRNA-mRNA pairs that correlate negatively or positively in expression level among cultivated rice. Genes involved in various metabolic processes and stress responses are enriched in the differentially expressed genes between rice indica and japonica subspecies. Our results indicate that stabilizing selection is the major force governing miRNA expression in cultivated rice, albeit positive selection may be responsible for much of the between-subspecies expression divergence. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  10. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.

    PubMed Central

    Kozak, M

    1984-01-01

    5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly. PMID:6694911

  11. Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

    PubMed

    Gale, Jenna R; Aschrafi, Armaz; Gioio, Anthony E; Kaplan, Barry B

    2017-06-01

    Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

  12. Does Lin28 Antagonize miRNA-Mediated Repression by Displacing miRISC from Target mRNAs?

    PubMed

    Kallen, Amanda N; Ma, Jing; Huang, Yingqun

    2012-01-01

    Lin28 is a developmentally regulated RNA-binding protein that plays important roles in diverse physiological and pathological processes including oncogenesis and brain synaptic function. These pleiotropic roles of Lin28 are mechanistically linked both to its ability to directly stimulate translation of genes involved primarily in cell growth and metabolism and to its ability to block biogenesis of a subset of miRNAs including the let-7 family of miRNAs. In the case of direct stimulation of gene expression, Lin28 binds to targeted mRNAs through recognition of Lin28-responsive elements (LREs) within mRNAs and recruits RNA helicase A (RHA) to promote translation. RHA belongs to the DEAD-box protein family of RNA helicases, which generally catalyze ATP-dependent unwinding of RNA duplexes or remodeling of ribonucleoprotein complexes (RNPs). Since any given mRNA can potentially be inhibited by miRNAs bearing complementary sequences, we hypothesize that binding of Lin28 to LREs not only nucleates the binding of multiple Lin28 molecules to the same mRNA, but also leads to remodeling of RNPs through recruitment of RHA and causes release of inhibitory miRNA-induced silencing complexes bound to the mRNA. This mode of action may contribute to Lin28-mediated stimulation of translation in both tumor and neuronal cells.

  13. PINK1 and Parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane.

    PubMed

    Gehrke, Stephan; Wu, Zhihao; Klinkenberg, Michael; Sun, Yaping; Auburger, Georg; Guo, Su; Lu, Bingwei

    2015-01-06

    Mitochondria play essential roles in many aspects of biology, and their dysfunction has been linked to diverse diseases. Central to mitochondrial function is oxidative phosphorylation (OXPHOS), accomplished by respiratory chain complexes (RCCs) encoded by nuclear and mitochondrial genomes. How RCC biogenesis is regulated in metazoans is poorly understood. Here we show that Parkinson's disease (PD)-associated genes PINK1 and Parkin direct localized translation of certain nuclear-encoded RCC (nRCC) mRNAs. Translationally repressed nRCC mRNAs are localized in a PINK1/Tom20-dependent manner to mitochondrial outer membrane, where they are derepressed and activated by PINK1/Parkin through displacement of translation repressors, including Pumilio and Glorund/hnRNP-F, a Parkin substrate, and enhanced binding of activators such as eIF4G. Inhibiting the translation repressors rescued nRCC mRNA translation and neuromuscular-degeneration phenotypes of PINK1 mutant, whereas inhibiting eIF4G had opposite effects. Our results reveal previously unknown functions of PINK1/Parkin in RNA metabolism and suggest new approaches to mitochondrial restoration and disease intervention.

  14. Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia

    SciTech Connect

    Weiner, D.M. Howard Hughes Medical Inst., Bethesda, MD ); Levey, A.I. Johns Hopkins Univ., Baltimore, MD ); Brann, M.R. )

    1990-09-01

    Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, the authors prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons; the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.

  15. Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

    PubMed Central

    Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

    2016-01-01

    The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

  16. Canonical Matches of Human MicroRNAs with mRNAs: A Broad Matrix of Position and Size.

    PubMed

    Parker, Michael S; Park, Edwards A; Sallee, Floyd R; Parker, Steven L

    2016-01-01

    Canonical hydrogen-bonding multi-nucleotide matches of microRNAs (miRs) with mRNAs are considered as important in mRNA regulation. MiR "seed" positions 2-8 are frequently viewed as mRNA partners, but there is ample evidence for use of other (and even non-contiguous) miR parts. No detailed information is available about canonical matching, and the GC content of the matches is rarely considered, although it should have a major regulatory potential. Sequences of 2586 human miRs and of 5'utr, cds and 3'utr in 18810 human mRNAs were examined for number and GC content of contiguous Watson-Crick antisense matches of six or more nucleotides (nt) in successive windows shifted by 1 nt. Frequency of the antisense matches is within all sectors similar for segments of up to 10 nt starting at positions 1-10 of miR sequences, with decrease of 3.5 to 4-fold for each 1-nt increment. Adenine and uracil rich elements (ARE-like) are very frequent in cds and 3'utr. All mRNAs have matches of up to 10 nt, and most also those of 11-15 nt. The match density is largest in 5'utr, and the match number in cds. The 5'utr and cds matches average much higher GC content than those of 3'utr. The GC content of matches is above that for the whole sector in 5'utr and cds, but lower in 3'utr. Human mRNA matches across miR sequences constitute a positionally similar matrix of canonical hydrogen-bonding reactivity. This presents ample opportunities for contiguous binding independent of miR position. The ubiquitous 10 to 15-nt matches could serve as binding foci. Interaction of miRs with the abundant GC-rich 5'utr and cds counterparts could be important in the regulation of mRNA-ribosome interaction as well as in mRNA disposal. The lower density and GC content of a majority of 3'utr matches could mainly support a dynamic regulation by miRs.

  17. Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

    PubMed

    Ávila-Mendoza, José; Pérez-Rueda, Ernesto; Urban-Sosa, Valeria; Carranza, Martha; Martínez-Moreno, Carlos G; Luna, Maricela; Arámburo, Carlos

    2017-09-30

    The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH2), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These

  18. Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae.

    PubMed Central

    Cross, S L; Smith, M M

    1988-01-01

    The haploid genome of Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 gene pairs, termed the copy I and copy II loci. The structures of the mRNA transcripts from each of these four genes were examined by nuclease protection and primer extension mapping. For each gene, several species of mRNAs were identified that differed in the lengths of their 5' and 3' untranslated regions. The cell cycle accumulation pattern of the H3 and H4 mRNAs was determined in cells from early-exponential-growth cultures fractionated by centrifugal elutriation. The RNA transcripts from all four genes were regulated with the cell division cycle, and transcripts from the nonallelic gene copies showed tight temporal coordination. Cell cycle regulation did not depend on selection of a particular histone mRNA transcript since the ratio of the multiple species from each gene remained the same across the division cycle. Quantitative measurements showed significant differences in the amounts of mRNA expressed from the two nonallelic gene sets. The mRNAs from the copy II H3 and H4 genes were five to seven times more abundant than the mRNAs from the copy I genes. There was no dosage compensation in the steady-state levels of mRNA when either set of genes was deleted. In particular, there was no increase in the amount of copy I H3 or H4 transcripts in cells in which the high-abundance copy II genes were deleted. Images PMID:3280973

  19. Isoform/variant mRNAs for sex steroid hormone receptors in humans.

    PubMed

    Hirata, Shuji; Shoda, Tomoko; Kato, Junzo; Hoshi, Kazuhiko

    2003-04-01

    The open reading frames of human sex steroid hormone receptors (hSSHRs) are composed of eight exons. In addition, the presence of various exons - including 5'-untranslated exons, alternative coding exons and novel 'intronic' exons - has been demonstrated in the genes encoding hSSHRs. The isoform/variant hSSHR mRNAs generated from thes e exons can be tentatively classified into seven types. In type 1, different mRNAs are generated with the use of alternative transcription start sites. In type 2, one or more exons are skipped. In type 3, one or more exons are duplicated. In type 4, distinct mRNAs containing different 5'-untranslated exon(s) are synthesized. In type 5, distinct mRNAs possessing different coding exon(s) are generated. In type 6, mRNA is synthesized by intronic exons and coding exons 4/5-8. In type 7, mRNA with insertion of intronic exon(s) is generated. Here, we review the isoform/variant hSSHR mRNAs and the structure of the genes encoding them.

  20. Promoter sequences direct cytoplasmic localization and translation of mRNAs during starvation in yeast.

    PubMed

    Zid, Brian M; O'Shea, Erin K

    2014-10-02

    A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes that encode proteins important for survival. Under many such conditions, the overall protein synthesis level is reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in Saccharomyces cerevisiae (yeast), translation is rapidly repressed, yet the transcription of many stress- and glucose-repressed genes is increased. Here we show, using ribosomal profiling and microscopy, that this transcriptionally upregulated gene set consists of two classes: one class produces messenger RNAs that are translated during glucose starvation and are diffusely localized in the cytoplasm, including many heat-shock protein mRNAs; and the other class produces mRNAs that are not efficiently translated during glucose starvation and are concentrated in foci that co-localize with P bodies and stress granules, a class that is enriched for mRNAs involved in glucose metabolism. Surprisingly, the information specifying the differential localization and protein production of these two classes of mRNA is encoded in the promoter sequence: promoter responsiveness to heat-shock factor 1 (Hsf1) specifies diffuse cytoplasmic localization and higher protein production on glucose starvation. Thus, promoter sequences can influence not only the levels of mRNAs but also the subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to the environmental conditions.

  1. Tissue specificity of epithelial keratins: differential expression of mRNAs from two multigene families.

    PubMed Central

    Kim, K H; Rheinwald, J G; Fuchs, E V

    1983-01-01

    Human epithelial cells cultured from stratified and simple squamous tissues all produce keratins of 40,000 to 58,000 daltons, but within this range the number and sizes vary with different epithelial cells. We have shown that this tissue-specific variation in the keratins is not due to posttranslational modification or processing, but rather to the differential expression of a family of heterogeneous but closely related mRNAs. All of these epithelial keratin mRNAs can be further grouped into two distinct subfamilies by their ability to hybridize with either of two cloned epidermal keratin cDNAs. All of the keratin mRNAs hybridize to one or the other, but not both, of the two cloned cDNAs. However, the mRNAs within each group hybridize with varying degrees of stringency, indicating that they are of similar but not identical sequence. Both types of keratin mRNAs are always expressed in every epithelial cell line studied, suggesting that filament assembly is dependent on the presence of both types of keratins. Within each of these two groups, the slight sequence differences in each class may reflect subtle tissue-specific variations in the structural and functional requirements of the epithelial cytoskeleton. Images PMID:6190074

  2. The role of deadenylation in the degradation of unstable mRNAs in trypanosomes

    PubMed Central

    Schwede, Angela; Manful, Theresa; Jha, Bhaskar Anand; Helbig, Claudia; Bercovich, Natalia; Stewart, Mhairi; Clayton, Christine

    2009-01-01

    Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5′–3′ exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5′ and 3′ ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA. PMID:19596809

  3. Different mRNAs code for dopa decarboxylase in tissues of neuronal and nonneuronal origin

    SciTech Connect

    Krieger, M.; Coge, F.; Gros, F.; Thibault, J. )

    1991-03-15

    A cDNA clone for dopa decarboxylase has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5{prime} end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Droxophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5{prime} untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3{prime} untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5{prime} untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.

  4. Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes

    PubMed Central

    1985-01-01

    The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48- h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h. The movement of all poly A+ mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A- reovirus mRNAs. The movement of the proteins encoded by the poly A+ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection. PMID:2858488

  5. Synthesis of full length cDNAs from four partially purified oviduct mRNAs.

    PubMed

    Buell, G N; Wickens, M P; Payvar, F; Schimke, R T

    1978-04-10

    Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each mRNA with the exception of conalbumin gives rise to a high proportion of full length cDNA. Several parameters previously reported to influence the size distribution of cDNA had no effect on the length of cDNA made from any mRNA fraction. The proportion of full length copy does depend on the reverse transcriptase lot.

  6. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs.

    PubMed

    Flint, S J; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.

  7. La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

    PubMed

    Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

    2017-04-07

    The 5'terminal oligopyrimidine (5'TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m(7)G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m(7)GTP), and a capped cytidine (m(7)GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

  8. Shwachman–Bodian–Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs

    PubMed Central

    In, Kyungmin; Zaini, Mohamad A.; Müller, Christine; Warren, Alan J.; von Lindern, Marieke; Calkhoven, Cornelis F.

    2016-01-01

    Mutations in the Shwachman–Bodian–Diamond Syndrome (SBDS) gene cause Shwachman–Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5′ untranslated regions (5′ UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype. PMID:26762974

  9. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs

    SciTech Connect

    Flint, S.J. . E-mail: sjflint@molbio.princeton.edu; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.

  10. Shwachman-Bodian-Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs.

    PubMed

    In, Kyungmin; Zaini, Mohamad A; Müller, Christine; Warren, Alan J; von Lindern, Marieke; Calkhoven, Cornelis F

    2016-05-19

    Mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene cause Shwachman-Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5' untranslated regions (5' UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Gene expression in cotton (Gossypium hirsutum L.) fiber: cloning of the mRNAs.

    PubMed Central

    John, M E; Crow, L J

    1992-01-01

    Cotton, an important natural fiber, is a differentiated epidermal cell. The number of genes that are active in fiber cells is similar to those in leaf, ovule, or root tissues. Through differential screening of a fiber cDNA library, we isolated five cDNA clones that are preferentially expressed in fiber. One of the cDNA clones, pCKE6, corresponded to an abundant mRNA in fiber. Transcripts for E6 were detected throughout the development of the fiber. Immunoprecipitation of in vitro translation products and Western blot analysis of fiber proteins showed two polypeptides in the range of 30-32 kDa as the products of E6 mRNA. Sequence analysis and hybrid-selected RNA translation also suggest that E6 mRNAs encode two polypeptides. Concentrations of E6 mRNA and protein are highest during the late primary cell wall and early secondary cell wall synthesis stages. Sequence comparison of E6 with other known eukaryotic and prokaryotic genes reveals no significant homology (GenBank; December 1991). E6 or a homologous gene(s) is conserved in several members of Malvaceae as well as in one other fiber-producing plant, kapok, but is not found in several other plants examined or in Acetobacter xylinum. A genomic clone corresponding to pCKE6 was isolated, and the promoter element of the E6 gene was shown to direct the expression of a carrot extensin mRNA in a tissue-specific and developmentally regulated fashion in transgenic cotton plants. Images PMID:1631059

  12. Gene expression in cotton (Gossypium hirsutum L.) fiber: cloning of the mRNAs.

    PubMed

    John, M E; Crow, L J

    1992-07-01

    Cotton, an important natural fiber, is a differentiated epidermal cell. The number of genes that are active in fiber cells is similar to those in leaf, ovule, or root tissues. Through differential screening of a fiber cDNA library, we isolated five cDNA clones that are preferentially expressed in fiber. One of the cDNA clones, pCKE6, corresponded to an abundant mRNA in fiber. Transcripts for E6 were detected throughout the development of the fiber. Immunoprecipitation of in vitro translation products and Western blot analysis of fiber proteins showed two polypeptides in the range of 30-32 kDa as the products of E6 mRNA. Sequence analysis and hybrid-selected RNA translation also suggest that E6 mRNAs encode two polypeptides. Concentrations of E6 mRNA and protein are highest during the late primary cell wall and early secondary cell wall synthesis stages. Sequence comparison of E6 with other known eukaryotic and prokaryotic genes reveals no significant homology (GenBank; December 1991). E6 or a homologous gene(s) is conserved in several members of Malvaceae as well as in one other fiber-producing plant, kapok, but is not found in several other plants examined or in Acetobacter xylinum. A genomic clone corresponding to pCKE6 was isolated, and the promoter element of the E6 gene was shown to direct the expression of a carrot extensin mRNA in a tissue-specific and developmentally regulated fashion in transgenic cotton plants.

  13. Single particle imaging of mRNAs crossing the nuclear pore: Surfing on the edge.

    PubMed

    Palazzo, Alexander F; Truong, Mathew

    2016-08-01

    Six years ago, the Singer lab published a landmark paper which described how individual mRNA particles cross the nuclear pore complex in mammalian tissue culture cells. This involved the simultaneous imaging of mRNAs, each labeled by a large number of tethered fluorescent proteins and fluorescently tagged nuclear pore components. Now two groups have applied this technique to the budding yeast Saccharomyces cerevisiae. Their results indicate that in the course of nuclear export, mRNAs likely engage complexes that are present on either side of the pore and that these interactions are modulated by proteins present in the messenger ribonucleoprotein (mRNP) complex. These findings lend support to the notion that just before and/or after the completion of nuclear export, mRNPs undergo one or more maturation steps that prepare the packaged mRNAs for translation. These results represent new and exciting insights into the mechanism of mRNA nuclear export.

  14. Translation dynamics of single mRNAs in live cells and neurons.

    PubMed

    Wu, Bin; Eliscovich, Carolina; Yoon, Young J; Singer, Robert H

    2016-06-17

    Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display "bursting" translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells.

  15. Characterization and Complexity of Wheat Developing Endosperm mRNAs 1

    PubMed Central

    Pernollet, Jean-Claude; Vaillant, Victor

    1984-01-01

    Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA+ RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)+ RNAs, analyzed on isokinetic sucrose gradient with [3H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly ⅔ and ⅓ of the MB poly(A)+ RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins. Images Fig. 2 PMID:16663795

  16. Cloning and characterization of the bovine class 1 and class 2 insulin-like growth factor-I mRNAs.

    PubMed

    Wang, Y; Price, S E; Jiang, H

    2003-11-01

    Insulin-like growth factor-I (IGF-I) is an important regulator of growth, development, and metabolism, and is the primary mediator of the growth-promoting activity of growth hormone (GH) in animals. In several species, the IGF-I polypeptide is generated from IGF-I mRNA containing either exon 1 (class 1 IGF-I mRNA) or exon 2 (class 2 IGF-I mRNA) as the leader exon. The objectives of this study were to identify class 1 and class 2 IGF-I mRNAs in cattle and to compare their expression in different tissues, their response to GH, and their translational efficiency. Three class 1 IGF-I cDNAs corresponding to three different transcription start sites in exon 1 and one class 2 IGF-I cDNA were identified from adult cattle liver using 5'-rapid amplification of cDNA ends (5'-RACE). Both classes of IGF-I mRNAs were expressed in a variety of tissues, with the highest level in liver; class 1 IGF-I mRNA was more abundant than class 2 IGF-I mRNA in all tissues. Six hours after a single intramuscular injection of 500 mg of recombinant bovine GH, class 1 and class 2 IGF-I mRNAs in steer liver were increased by 29% (P=0.07) and 62% (P<0.05), respectively. The luciferase reporter mRNA fused to a class 1 IGF-I 5'-untranslated region (5'-UTR) was translated four times more efficiently in vitro than the luciferase reporter mRNA fused to a class 2 IGF-I 5'-UTR (P<0.05). These results indicate that the IGF-I gene in cattle is transcribed as class 1 and class 2 IGF-I mRNAs and that the two classes of IGF-I mRNAs may be regulated differentially at both transcriptional and translational levels.

  17. Labeling of target mRNAs using a photo-reactive microRNA probe.

    PubMed

    Nakamoto, Kosuke; Minami, Koichiro; Akao, Yukihiro; Ueno, Yoshihito

    2016-05-10

    To identify target mRNAs of an miRNA, we synthesized photo-reactive miRNA probes, which contained a photo-reactive nucleoside analog, 1-O-[4-(3-trifluoromethyl-3H-diazirine-3-yl)]benzyl-β-d-ribofuranose, in the middle of the strand. The photo-reactive miRNA-145 probe was found to specifically label the target mRNAs, FSCN1 and KLF4, by UV-A irradiation in human colon cancer DLD-1 cells.

  18. Messengers, motors and mysteries: sorting of eukaryotic mRNAs by cytoskeletal transport.

    PubMed

    Bullock, Simon L

    2011-10-01

    It has become increasingly apparent in recent years that the subcellular localization of specific mRNAs is a prevalent method for spatially controlling gene expression. In most cases, targeting of mRNAs is mediated by transport along cytoskeletal filaments by molecular motors. However, the means by which specific messages are recognized and linked to the motors are poorly understood. Here, I will provide an overview of recent progress in elucidating the molecular mechanisms and principles of mRNA transport, including several studies highlighting the co-operation of different motors during the localization process. Important outstanding questions will also be highlighted.

  19. Isolation and functional property of mRNAs coding for human interleukin 2.

    PubMed

    Beck, H P; Schneider, E M; Wallenfels, B; Lipps, H J; Schott, H; Sonneborn, H; Wernet, P

    1984-03-01

    Interleukin 2 (IL 2) is a lymphokine with specific growth promoting properties for activated T cells. The isolation and purification of specific mRNAs coding for this protein is reported. The analysis of total poly A + containing RNA from PHA stimulated T lymphocytes on agarose gels reveals the existence of at least two different IL-2 mRNAs. Isolated RNAs have been microinjected separately into Xenopus laevis oocytes for translation and consecutively the synthesized proteins were tested for activity on alloactivated T cell lines which were dependent on IL-2 for growth. Both RNAs coded for a protein which displayed clearly detectable IL-2 activity.

  20. Modification of Selenoprotein mRNAs by Cap Tri-methylation.

    PubMed

    Gribling-Burrer, Anne-Sophie; Eriani, Gilbert; Allmang, Christine

    2018-01-01

    Several selenoprotein mRNAs undergo 5' cap maturation events whereby their classical monomethylated m(7)G cap becomes trimethylated (m3(2,2,7)G) by the trimethylguanosine synthase 1 (Tgs1). Here, we describe immunoprecipitation methods for the detection of endogenous m3(2,2,7)G-capped selenoprotein mRNAs from total cell extracts or after polysome fractionation of cytoplasmic extracts. We have also developed a method for the in vitro cap hypermethylation of selenoprotein mRNA transcripts using purified Tgs1 enzyme.

  1. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation*

    PubMed Central

    Timpano, Sara; Uniacke, James

    2016-01-01

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5′ cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or “normoxia,” is far from physiological or “normal.” In fact, oxygen in human tissues ranges from 1–11% or “physioxia.” Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1–11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. PMID:27002144

  2. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation.

    PubMed

    Timpano, Sara; Uniacke, James

    2016-05-13

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin*

    PubMed Central

    Korneeva, Nadejda L.; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E.

    2016-01-01

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5′-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5′-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357–1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m7GTP-Sepharose reveals that both CGP and eIF4G(1357–1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5′-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. PMID:26668315

  4. Adaptation of a corticosterone ELISA to demonstrate sequence-specific effects of angiotensin II peptides and C-type natriuretic peptide on 1alpha-hydroxycorticosterone synthesis and steroidogenic mRNAs in the elasmobranch interrenal gland.

    PubMed

    Evans, Andrew N; Rimoldi, John M; Gadepalli, Rama S V; Nunez, B Scott

    2010-06-01

    It is thought that a single corticosteroid, 1alpha-hydroxycorticosterone (1alpha-B), is both a glucocorticoid and mineralocorticoid in the elasmobranch fishes. We investigated the putative mineralocorticoid role of 1alpha-B by examining regulation of interrenal 1alpha-B synthesis by osmoregulatory hormones in the euryhaline stingray Dasyatis sabina. Using synthesized steroid, a commercial enzyme-linked immunoassay was validated for the quantification of 1alpha-B. In interrenal cultures, the antinatriuretic peptide angiotensin II (ANG II) was potently steroidogenic, whereas C-type natriuretic peptide had no effect on 1alpha-B titers. However, both peptides significantly decreased abundance of rate-limiting steroidogenic mRNAs (steroidogenic acute regulatory protein, StAR; cholesterol side-chain cleavage, P450scc). We also isolated cDNAs encoding ANG II from three species of elasmobranch, verifying heterogeneity among elasmobranch peptides at the first amino acid position. Potential implications of this heterogeneity were investigated by examining the effects of homologous and heterologous ANG II on interrenal steroid production and steroidogenic mRNAs. Changes at amino acid position three, but not position one, of ANG II significantly affected steroidogenic potency. Conversely, changes at position one, but not position three, significantly affected the potency of ANG II to alter levels of steroidogenic mRNAs. This study is the first to demonstrate regulation of elasmobranch steroidogenic mRNAs by osmoregulatory peptides. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. Zar1 represses translation in Xenopus oocytes and binds to the TCS in maternal mRNAs with different characteristics than Zar2.

    PubMed

    Yamamoto, Tomomi M; Cook, Jonathan M; Kotter, Cassandra V; Khat, Terry; Silva, Kevin D; Ferreyros, Michael; Holt, Justin W; Knight, Jefferson D; Charlesworth, Amanda

    2013-10-01

    Maternal mRNAs are translationally regulated during early development. Zar1 and its closely related homolog, Zar2, are both crucial in early development. Xenopus laevis Zygote arrest 2 (Zar2) binds to the Translational Control Sequence (TCS) in maternal mRNAs and regulates translation. The molecular mechanism of Zar1 has not been described. Here we report similarities and differences between Xenopus Zar1 and Zar2. Analysis of Zar sequences in vertebrates revealed two Zar family members with conserved, characteristic amino acid differences in the C-terminal domain. The presence of only two vertebrate Zar proteins was supported by analyzing Zar1 synteny. We propose that the criteria for naming Zar sequences are based on the characteristic amino acids and the chromosomal context. We also propose reclassification of some Zar sequences. We found that Zar1 is expressed throughout oogenesis and is stable during oocyte maturation. The N-terminal domain of Zar1 repressed translation of a reporter construct in immature oocytes. Both Zar1 and Zar2 bound to the TCS in the Wee1 and Mos 3' UTRs using a zinc finger in the C-terminal domain. However, Zar1 had much higher affinity for RNA than Zar2. To show the functional significance of the conserved amino acid substitutions, these residues in Zar2 were mutated to those found in Zar1. We show that these residues contributed to the different RNA binding characteristics of Zar1 compared to Zar2. Our study shows that Zar proteins have generally similar molecular functions in the translational regulation of maternal mRNAs, but they may have different roles in early development.

  6. Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues

    PubMed Central

    Schaik, R H N van; Wierikx, C D J; Timmerman, M A; Oomen, M H; Weerden, W M van; Kwast, T H van der; Steenbrugge, G J van; Jong, F H de

    1999-01-01

    The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate pithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour aterial when compared to non-malignant tissue (P< 0.05; Mann–Whitney U -test). Inhibin/activin βA- and βB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of βB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While βB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin α-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin βB-subunit mRNA or by a decrease of ActRIB mRNA levels. © 2000 Cancer Research Campaign PMID:10638976

  7. Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs

    PubMed Central

    2014-01-01

    Background MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5′ end of the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs. Results We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artifact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions. Conclusions Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence. PMID:24629056

  8. The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

    PubMed

    Dauber, Bianca; Saffran, Holly A; Smiley, James R

    2014-09-01

    We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during

  9. Characteristics of glycine receptors expressed by embryonic rat brain mRNAs

    PubMed Central

    García-Alcocer, Guadalupe; García-Colunga, Jesús; Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2001-01-01

    A study was made of glycine (Gly) and γ-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 ± 35, 346 ± 28, and 389 ± 22 nA, whereas the GABA-currents (1 mM GABA) were all ≤40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC50 for Gly was 611 ± 77 μM for encephalon, 661 ± 28 μM for midbrain, and 506 ± 18 μM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all ≈2. Strychnine inhibited all of the Gly-currents, with an IC50 of 56 ± 3 nM for encephalon, 97 ± 4 nM for midbrain, and 72 ± 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca2+ concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription–PCR studies showed differences in the Gly receptor (GlyR) α-subunits expressed, whereas the β-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca2+ could play important functions during brain development. PMID:11226317

  10. Method for the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts using RIP-Chip.

    PubMed

    Dahm, Garrett M; Gubin, Matthew M; Magee, Joseph D; Techasintana, Patsharaporn; Calaluce, Robert; Atasoy, Ulus

    2012-09-29

    As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

  11. Correlated mRNAs and miRNAs from co-expression and regulatory networks affect porcine muscle and finally meat properties.

    PubMed

    Ponsuksili, Siriluck; Du, Yang; Hadlich, Frieder; Siengdee, Puntita; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus

    2013-08-05

    Physiological processes aiding the conversion of muscle to meat involve many genes associated with muscle structure and metabolic processes. MicroRNAs regulate networks of genes to orchestrate cellular functions, in turn regulating phenotypes. We applied weighted gene co-expression network analysis to identify co-expression modules that correlated to meat quality phenotypes and were highly enriched for genes involved in glucose metabolism, response to wounding, mitochondrial ribosome, mitochondrion, and extracellular matrix. Negative correlation of miRNA with mRNA and target prediction were used to select transcripts out of the modules of trait-associated mRNAs to further identify those genes that are correlated with post mortem traits. Porcine muscle co-expression transcript networks that correlated to post mortem traits were identified. The integration of miRNA and mRNA expression analyses, as well as network analysis, enabled us to interpret the differentially-regulated genes from a systems perspective. Linking co-expression networks of transcripts and hierarchically organized pairs of miRNAs and mRNAs to meat properties yields new insight into several biological pathways underlying phenotype differences. These pathways may also be diagnostic for many myopathies, which are accompanied by deficient nutrient and oxygen supply of muscle fibers.

  12. Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice.

    PubMed

    Xie, Fang; Anderson, Christopher L; Timme, Kelsey R; Kurz, Scott G; Fernando, Samodha C; Wood, Jennifer R

    2016-04-01

    RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.

  13. Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice

    PubMed Central

    Xie, Fang; Anderson, Christopher L.; Timme, Kelsey R.; Kurz, Scott G.; Fernando, Samodha C.

    2016-01-01

    RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients. PMID:26881311

  14. Altered expression of retinoic acid (RA) receptor mRNAs in the fetal mouse secondary palate by all-trans and 13-cis RAs: implications for RA-induced teratogenesis.

    PubMed

    Naitoh, H; Mori, C; Nishimura, Y; Shiota, K

    1998-01-01

    Retinoic acid (RA) is mandatory for various biological processes and normal embryonic development but is teratogenic at high concentrations. In rodents, one of the major malformations induced by RA is cleft palate (CP). RA mediates its effects by RA receptors (RARs), but the expression patterns of RARs in the developing palate are still unclear. We investigated the normal expression of RAR alpha, beta, and gamma messenger RNAs (mRNAs) in the fetal mouse secondary palate and the effects of all-trans and 13-cis RAs on the expression of RAR mRNAs by Northern blot analysis. RAR alpha (2.8, 3.8 kb), RAR beta (3.3 kb), and RAR gamma (3.7 kb) mRNAs were detected in the fetal palate on gestational days (GD) 12.5-14.5. The expression of RAR alpha and gamma mRNAs did not show apparent sequential changes, but that of RAR beta mRNA increased at GD 13.5. Treatment of pregnant mice with 100 mg/kg all-trans RA induced CP in 94% of the fetuses and elevated the levels of RAR beta and gamma mRNAs in the fetal palate. The up-regulation of RAR beta mRNA by all-trans RA was more marked than that of RAR gamma mRNA. Treatment with 100 mg/kg 13-cis RA induced CP in only 19% of the fetuses. Although 13-cis RA elevated the RAR beta and gamma mRNA levels in fetal palates, its up-regulation was slower and less marked than that induced by all-trans RA. These findings indicate that the induction of RAR beta mRNA in the fetal palate correlates well with the tissue concentration of all-trans RA after RA treatment, and RAR beta may be one of the most influential candidate molecules for RA-induced teratogenesis.

  15. Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens.

    PubMed

    Morton, Matthew L; Bai, Xiaodong; Merry, Callie R; Linden, Philip A; Khalil, Ahmad M; Leidner, Rom S; Thompson, Cheryl L

    2014-07-01

    Adenocarcinoma in situ (AIS) is an intermediate step in the progression of normal lung tissue to invasive adenocarcinoma. However, molecular mechanisms underlying this progression remain to be fully elucidated due to challenges in obtaining fresh clinical samples for downstream analyses. Formalin fixation and paraffin embedding (FFPE) is a tissue preservation system widely used for long-term storage. Until recently, challenges in working with FFPE precluded using new RNA sequencing technologies (RNA-seq), which would help clarify key pathways in cancer progression. Also, isolation techniques including laser-capture micro-dissection provide the ability to select histopathologically distinct tissues, allowing researchers to study transcriptional variations between tightly juxtaposed cell and tissue types. Utilizing these technologies and new alignment tools we examined differential expression of long intergenic non-coding RNAs (lincRNAs) and mRNAs across normal, AIS and invasive adenocarcinoma samples from six patients to identify possible markers of lung cancer progression. RNA extracted and sequenced from these 18 samples generated an average of 198 million reads per sample. After alignment and filtering, uniquely aligned reads represented an average 35% of the total reads. We detected differential expression of a number of lincRNAs and mRNAs when comparing normal to AIS, or AIS to invasive adenocarcinoma. Of these, 5 lincRNAs and 31 mRNAs were consistently up- or down-regulated from normal to AIS and more so to invasive carcinoma. We validated the up-regulation of two mRNAs and one lincRNA by RT-qPCR as proof of principle. Our findings indicate a potential role of not only mRNAs, but also lincRNAs in the progression to invasive adenocarcinoma. We anticipate that these findings will lay the groundwork for future experimental studies of candidate RNAs from FFPE to identify their functional roles in lung cancer. Copyright © 2014 Elsevier Ireland Ltd. All rights

  16. Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1.

    PubMed

    Pimentel, Belén; Madine, Mark A; de la Cueva-Méndez, Guillermo

    2005-10-05

    Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection.

  17. DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs

    PubMed Central

    Yamaji, Masashi; Jishage, Miki; Meyer, Cindy; Suryawanshi, Hemant; Der, Evan; Yamaji, Misaki; Garzia, Aitor; Morozov, Pavel; Manickavel, Sudhir; McFarland, Hannah L.; Roeder, Robert G.; Hafner, Markus; Tuschl, Thomas

    2017-01-01

    The vertebrate-conserved RNA-binding protein (RBP) DND1 is required for survival of primordial germ cells (PGCs), as well as germ cell tumour (TGCT) suppression in mice1–5. Here we report that DND1 binds a UU[A/U] trinucleotide motif predominantly in messenger RNA (mRNA) 3′ untranslated regions (UTRs), and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase (CCR4) complex. Transcriptomic analysis revealed that the extent of suppression is dependent on the number of DND1 binding sites. The DND1-dependent mRNA destabilization is required for survival of murine PGCs and spermatogonial stem cells (SSCs) by suppressing apoptosis. The target RNA spectrum includes positive regulators of apoptosis, inflammation, and modulators of signalling pathways regulating stem cell pluripotency including the TGF-β super family, all of which are aberrantly elevated in Dnd1-deficient PGCs. We propose that the induction of the posttranscriptional suppressor DND1 synergizes with concurrent transcriptional changes to sharpen developmental transitions during cellular differentiation and maintenance of the germline. PMID:28297718

  18. A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs.

    PubMed

    Sebastian, Sarah; Brochet, Mathieu; Collins, Mark O; Schwach, Frank; Jones, Matthew L; Goulding, David; Rayner, Julian C; Choudhary, Jyoti S; Billker, Oliver

    2012-07-19

    Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3' and 5'UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. A Plasmodium Calcium-Dependent Protein Kinase Controls Zygote Development and Transmission by Translationally Activating Repressed mRNAs

    PubMed Central

    Sebastian, Sarah; Brochet, Mathieu; Collins, Mark O.; Schwach, Frank; Jones, Matthew L.; Goulding, David; Rayner, Julian C.; Choudhary, Jyoti S.; Billker, Oliver

    2012-01-01

    Summary Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3′ and 5′UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression. PMID:22817984

  20. DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs.

    PubMed

    Yamaji, Masashi; Jishage, Miki; Meyer, Cindy; Suryawanshi, Hemant; Der, Evan; Yamaji, Misaki; Garzia, Aitor; Morozov, Pavel; Manickavel, Sudhir; McFarland, Hannah L; Roeder, Robert G; Hafner, Markus; Tuschl, Thomas

    2017-03-23

    The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFβ superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.

  1. Expression of mRNAs coding for the alpha 1 chain of type XIII collagen in human fetal tissues: comparison with expression of mRNAs for collagen types I, II, and III

    PubMed Central

    1989-01-01

    This paper describes the topographic distribution of the multiple mRNAs coding for a novel human short-chain collagen, the alpha 1 chain of type XIII collagen. To identify the tissues and cells expressing these mRNAs, human fetal tissues of 15-19 gestational wk were studied by Northern and in situ hybridizations. The distribution pattern of the type XIII collagen mRNAs was compared with that of fibrillar collagen types I, II, and III using specific human cDNA probes for each collagen type. Northern hybridization showed the bone, cartilage, intestine, skin, and striated muscle to contain mRNAs for type XIII collagen. An intense in situ hybridization signal was obtained with the type XIII collagen cDNAs in the epidermis, hair follicles, and nail root cells of the skin, whereas the fibrillar collagen mRNAs were detected in the dermis. Cells in the intestinal mucosal layer also appeared to contain high levels of alpha 1(XIII) collagen mRNAs, but contained none of the fibrillar collagen mRNAs. In the bone and striated muscle, alpha 1(XIII) collagen mRNAs were detected in the mesenchymal cells forming the reticulin fibers of the bone marrow and endomycium. The hybridization signal obtained with the alpha 1(XIII) collagen cDNA probe in cartilaginous areas of the growth plates was similar, but less intense, to that obtained with the type II collagen probe. A clear hybridization signal was also detected at the (pre)articular surfaces and at the margins of the epiphyses, whereas it was weaker in the resting chondrocytes in the middle of the epiphyses. The brain, heart, kidney, liver, lung, placenta, spleen, testis, tendon, and thymus did not appear to contain alpha 1(XIII) collagen mRNAs. PMID:2768343

  2. A cell type-specific view on the translation of mRNAs from ROS-responsive genes upon paraquat treatment of Arabidopsis thaliana leaves.

    PubMed

    Benina, Maria; Ribeiro, Dimas Mendes; Gechev, Tsanko S; Mueller-Roeber, Bernd; Schippers, Jos H M

    2015-02-01

    Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5'-untranslated region (5'-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress.

  3. Co-expression of alpha7 and beta2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons.

    PubMed

    Azam, L; Winzer-Serhan, U; Leslie, F M

    2003-01-01

    Nicotine enhances cognitive and attentional processes through stimulation of the basal forebrain cholinergic system. Although muscarinic cholinergic autoreceptors have been well characterized, pharmacological characterization of nicotinic autoreceptors has proven more difficult. The present study used double-labeling in situ hybridization to determine expression of nicotinic acetylcholine receptor (nAChR) subunit mRNAs within basal forebrain cholinergic neurons in order to gain information about possible nAChR autoreceptor properties. Cholinergic cells of the mesopontine tegmentum and striatal interneurons were also examined, as were septohippocampal GABAergic neurons that interact with cholinergic neurons to regulate hippocampal activity. alpha7 and beta2 nAChR mRNAs were found to be co-expressed in almost all cholinergic cells and in the majority of GABAergic neurons examined. alpha4 nAChR mRNA expression was restricted to cholinergic cells of the nucleus basalis magnocellularis, and to non-cholinergic cells of the medial septum and mesopontine tegmentum. These data suggest possible regional differences in the pharmacological properties of nicotinic autoreceptors on cholinergic cells. Whereas most cholinergic cells express rapidly desensitizing alpha7 homomers or alpha7beta2 heteromers, cortical projection neurons may also express a pharmacologically distinct alpha4beta2 nAChR subtype. There may also be differential nAChR regulation of cholinergic and non-cholinergic cells within the mesopontine tegmentum that are implicated in acquisition of nicotine self-administration.

  4. The 3'-terminal region of the mRNAs for VSG and procyclin can confer stage specificity to gene expression in Trypanosoma brucei.

    PubMed

    Berberof, M; Vanhamme, L; Tebabi, P; Pays, A; Jefferies, D; Welburn, S; Pays, E

    1995-06-15

    The variant surface glycoprotein (VSG) and procyclin are the respective major surface antigens of the bloodstream and the procyclic forms of Trypanosoma brucei. These proteins and their mRNAs are both the most abundant and absolutely characteristic of their respective life cycle stages. We show that the 3'-terminal region of these mRNAs regulates expression of a reporter gene in an inverse manner, depending on the developmental form of the parasite. In the case of VSG mRNA, the 97 nt sequence upstream from the polyadenylation site is responsible for these effects. The regulation occurs through a variation of mRNA abundance which is not due to a change in primary transcription. In the bloodstream form this effect is manifested by an increase in RNA stability, whereas in the procyclic form it seems to be related to a reduction in the efficiency of mRNA maturation. The 3'-end of VSG mRNA can obviate the 5- to 10-fold stimulation of transcription driven by the procyclin promoter during differentiation from the bloodstream to the procyclic form. The predominance of posttranscriptional over transcriptional controls is probably linked to the organization of the trypanosome genome in polycistronic transcription units.

  5. Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells

    PubMed Central

    Ghildiyal, Megha; Seitz, Hervé; Horwich, Michael D.; Li, Chengjian; Du, Tingting; Lee, Soohyun; Xu, Jia; Kittler, Ellen L.W.; Zapp, Maria L.; Weng, Zhiping; Zamore, Phillip D.

    2009-01-01

    Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line. PMID:18403677

  6. Synthetic Capped mRNAs for Cap-Specific Photo-Cross-Linking Experiments.

    PubMed

    Kowalska, Joanna; Martin, Franck; Jemielity, Jacek

    2016-01-01

    The 7-methylguanosine triphosphate cap present at the 5' ends of eukaryotic mRNAs plays numerous roles in mRNA expression and metabolism. The identification and studies on cap-binding partners can be significantly advanced using tailored chemical tools such as synthetic cap analogues or RNAs carrying modified cap structures. Here we provide protocols for the production of mRNAs specifically labeled within the 5' cap with a nucleoside capable of being photo-activated, either 6-thioguanosine or 7-methyl-6-thioguanosine, which can be used in photo-cross-linking experiments to identify or characterize cap-binding biomolecules. We also describe a protocol for the cross-linking experiments with capped RNAs to map histone H4 cap-binding pocket.

  7. Urine podocyte mRNAs mark disease activity in IgA nephropathy.

    PubMed

    Fukuda, Akihiro; Sato, Yuji; Iwakiri, Takashi; Komatsu, Hiroyuki; Kikuchi, Masao; Kitamura, Kazuo; Wiggins, Roger C; Fujimoto, Shouichi

    2015-07-01

    Podocyte depletion is a major mechanism driving glomerulosclerosis. We and others have previously projected from model systems that podocyte-specific mRNAs in the urine pellet might serve as glomerular disease markers. We evaluated IgA nephropathy (IgAN) to test this concept. From 2009 to 2013, early morning voided urine samples and kidney biopsies from IgAN patients (n = 67) were evaluated in comparison with urine samples from healthy age-matched volunteers (n = 28). Urine podocyte (podocin) mRNA expressed in relation to either urine creatinine concentration or a kidney tubular marker (aquaporin 2) was tested as markers. Urine podocyte mRNAs were correlated with the severity of active glomerular lesions (segmental glomerulosclerosis and acute extracapillary proliferation), but not with non-glomerular lesions (tubular atrophy/interstitial fibrosis) or with clinical parameters of kidney injury (serum creatinine and estimated glomerular filtration rate), or with degree of accumulated podocyte loss at the time of biopsy. In contrast, proteinuria correlated with all histological and clinical markers. Glomerular tuft podocyte nuclear density (a measure of cumulative podocyte loss) correlated with tubular atrophy/interstitial fibrosis, estimated-glomerular filtration rate and proteinuria, but not with urine podocyte markers. In a subset of the IgA cohort (n = 19, median follow-up period = 37 months), urine podocyte mRNAs were significantly decreased after treatment, in contrast to proteinuria which was not significantly changed. Urine podocyte mRNAs reflect active glomerular injury at a given point in time, and therefore provide both different and additional clinical information that can complement proteinuria in the IgAN decision-making paradigm. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

  8. Mutant LYS2 mRNAs retained and degraded in the nucleus of Saccharomyces cerevisiae

    PubMed Central

    Das, Biswadip; Das, Satarupa; Sherman, Fred

    2006-01-01

    We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Δ or cbc2-Δ mutants lacking components of the cap-binding complex and in rrp6-Δ mutants lacking Rrp6p, a 3′ to 5′ nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Δ and rrp6-Δ. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon. LYS2 mRNAs from the lys2-187 and related mutants were rapidly degraded, and the degradation was suppressed by cbc1-Δ and rrp6-Δ. The U1A-GFP imaging procedure was used to show that the lys2-187 mRNA was partially retained in the nucleus, explaining the susceptibility to DRN. The creation of several derivatives of lys2-187 by site-directed mutagenesis revealed that the in-frame TGA by itself was not responsible for the increased susceptibility to DRN. Thus, mRNAs susceptible to DRN can be formed by a 2-bp change. Furthermore, this “retention signal” causing susceptibility to DRN is lost by altering one of the base pairs, establishing that mRNAs susceptible and unsusceptible to DRN can be attributed to a single nucleotide in the proper context. PMID:16832048

  9. Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons.

    PubMed

    Gervasi, Christine; Thyagarajan, Amar; Szaro, Ben G

    2003-06-23

    Characteristic changes in the expression of neuronal intermediate filaments (nIFs), an abundant cytoskeletal component of vertebrate axons, accompany successful axon regeneration. In mammalian regenerating PNS, expression of nIFs that are characteristic of mature neurons becomes suppressed throughout regeneration, whereas that of peripherin, which is abundant in developing axons, increases. Comparable changes are absent from mammalian injured CNS; but in goldfish and lamprey CNS, expression of several nIFs increases during axon regrowth. To obtain a broader view of the nIF response of successfully regenerating vertebrate CNS, in situ hybridization and video densitometry were used to track multiple nIF mRNAs during optic axon regeneration in Xenopus laevis. As in other successfully regenerating systems, peripherin expression increased rapidly after injury and expression of those nIFs characteristic of mature retinal ganglion cells decreased. Unlike the decrease in nIF mRNAs of regenerating PNS, that of Xenopus retinal ganglion cells was transient, with most nIF mRNAs increasing above normal during axon regrowth. At the peak of regeneration, increases in each nIF mRNA resulted in a doubling of the total amount of nIF mRNA, as well as a shift in the relative proportions contributed by each nIF. The relative proportions of peripherin and NF-M increased above normal, whereas proportions of xefiltin and NF-L decreased and that of XNIF remained the same. The increases in peripherin and NF-M mRNAs were accompanied by increases in protein. These results are consistent with the hypothesis that successful axon regeneration involves changes in nIF subunit composition conducive to growth and argue that a successful injury response differs between CNS and PNS. Copyright 2003 Wiley-Liss, Inc.

  10. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm

    PubMed Central

    Vrettos, Nicholas; Maragkakis, Manolis; Mourelatos, Zissimos

    2016-01-01

    The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) play a central role in genomic stability, which is inextricably tied with germ cell formation, by forming ribonucleoproteins (piRNPs) that silence transposable elements (TEs)1. In Drosophila melanogaster and other animals, primordial germ cell (PGC) specification in the developing embryo is driven by maternal mRNAs and proteins that assemble into specialized mRNPs localized in the germ (pole) plasm at the posterior of the oocyte2,3. Maternal piRNPs, especially those loaded on Aubergine (Aub), a Piwi protein, are transmitted to the germ plasm to initiate transposon silencing in the offspring germline4–7. Transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process8; mRNAs necessary for PGC formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, whose molecular identity remains unknown8,9. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm10–12 and interactions between Aub and Tudor are essential for the formation of germ granules13–16. Here we show that Aub-loaded piRNAs use partial base pairing characteristic of Argonaute RNPs to bind mRNAs randomly, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Strikingly, germ plasm mRNAs in Drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. Our findings reveal an unexpected function for Piwi ribonucleoprotein complexes in mRNA trapping that may be generally relevant to the function of animal germ granules. PMID:26950602

  11. Genome-wide analysis of mRNAs associated with mouse peroxisomes.

    PubMed

    Yarmishyn, Aliaksandr A; Kremenskoy, Maksym; Batagov, Arsen O; Preuss, Axel; Wong, Jin Huei; Kurochkin, Igor V

    2016-12-22

    RNA is often targeted to be localized to the specific subcellular compartments. Specific localization of mRNA is believed to be an important mechanism for targeting their protein products to the locations, where their function is required. In this study we performed the genome wide transcriptome analysis of peroxisome preparations from the mouse liver using microarrays. We demonstrate that RNA is absent inside peroxisomes, however it is associated at their exterior via the noncovalent contacts with the membrane proteins. We detect enrichment of specific sets of transcripts in two preparations of peroxisomes, purified with different degrees of stringency. Importantly, among these were mRNAs encoding bona fide peroxisomal proteins, such as peroxins and peroxisomal matrix enzymes involved in beta-oxidation of fatty acids and bile acid biosynthesis. The top-most enriched mRNA, whose association with peroxisomes we confirm microscopically was Hmgcs1, encoding 3-hydroxy-3-methylglutaryl-CoA synthase, a crucial enzyme of cholesterol biosynthesis pathway. We observed significant representation of mRNAs encoding mitochondrial and secreted proteins in the peroxisomal fractions. This is a pioneer genome-wide study of localization of mRNAs to peroxisomes that provides foundation for more detailed dissection of mechanisms of RNA targeting to subcellular compartments.

  12. Fragile X mental retardation protein FMRP binds mRNAs in the nucleus.

    PubMed

    Kim, Miri; Bellini, Michel; Ceman, Stephanie

    2009-01-01

    The fragile X mental retardation protein FMRP is an RNA binding protein that associates with a large collection of mRNAs. Since FMRP was previously shown to be a nucleocytoplasmic shuttling protein, we examined the hypothesis that FMRP binds its cargo mRNAs in the nucleus. The enhanced green fluorescent protein-tagged FMRP construct (EGFP-FMRP) expressed in Cos-7 cells was efficiently exported from the nucleus in the absence of its nuclear export sequence and in the presence of a strong nuclear localization sequence (the simian virus 40 [SV40] NLS), suggesting an efficient mechanism for nuclear export. We hypothesized that nuclear FMRP exits the nucleus through its bound mRNAs. Using silencing RNAs to the bulk mRNA exporter Tap/NXF1, we observed a significantly increased number of cells containing EGFP-FMRP in the nucleus, which was further augmented by removal of FMRP's nuclear export sequence. Nuclear-retained SV40-FMRP could be released upon treatment with RNase. Further, Tap/NXF1 coimmunoprecipitated with EGFP-FMRP in an RNA-dependent manner and contained the FMR1 mRNA. To determine whether FMRP binds pre-mRNAs cotranscriptionally, we expressed hemagglutinin-SV40 FMRP in amphibian oocytes and found it, as well as endogenous Xenopus FMRP, on the active transcription units of lampbrush chromosomes. Collectively, our data provide the first lines of evidence that FMRP binds mRNA in the nucleus.

  13. Predicting effective microRNA target sites in mammalian mRNAs

    PubMed Central

    Agarwal, Vikram; Bell, George W; Nam, Jin-Wu; Bartel, David P

    2015-01-01

    MicroRNA targets are often recognized through pairing between the miRNA seed region and complementary sites within target mRNAs, but not all of these canonical sites are equally effective, and both computational and in vivo UV-crosslinking approaches suggest that many mRNAs are targeted through non-canonical interactions. Here, we show that recently reported non-canonical sites do not mediate repression despite binding the miRNA, which indicates that the vast majority of functional sites are canonical. Accordingly, we developed an improved quantitative model of canonical targeting, using a compendium of experimental datasets that we pre-processed to minimize confounding biases. This model, which considers site type and another 14 features to predict the most effectively targeted mRNAs, performed significantly better than existing models and was as informative as the best high-throughput in vivo crosslinking approaches. It drives the latest version of TargetScan (v7.0; targetscan.org), thereby providing a valuable resource for placing miRNAs into gene-regulatory networks. DOI: http://dx.doi.org/10.7554/eLife.05005.001 PMID:26267216

  14. Stable Membrane-Association of mRNAs in Etiolated, Greening and Mature Plastids

    PubMed Central

    Legen, Julia; Schmitz-Linneweber, Christian

    2017-01-01

    Chloroplast genes are transcribed as polycistronic precursor RNAs that give rise to a multitude of processing products down to monocistronic forms. Translation of these mRNAs is realized by bacterial type 70S ribosomes. A larger fraction of these ribosomes is attached to chloroplast membranes. This study analyzed transcriptome-wide distribution of plastid mRNAs between soluble and membrane fractions of purified plastids using microarray analyses and validating RNA gel blot hybridizations. To determine the impact of light on mRNA localization, we used etioplasts, greening plastids and mature chloroplasts from Zea mays as a source for membrane and soluble extracts. The results show that the three plastid types display an almost identical distribution of RNAs between the two organellar fractions, which is confirmed by quantitative RNA gel blot analyses. Furthermore, they reveal that different RNAs processed from polycistronic precursors show transcript-autonomous distribution between stroma and membrane fractions. Disruption of ribosomes leads to release of mRNAs from membranes, demonstrating that attachment is likely a direct consequence of translation. We conclude that plastid mRNA distribution is a stable feature of different plastid types, setting up rapid chloroplast translation in any plastid type. PMID:28858216

  15. An "up, no change, or down" system: Time-dependent expression of mRNAs in contused skeletal muscle of rats used for wound age estimation.

    PubMed

    Sun, Jun-Hong; Zhu, Xi-Yan; Dong, Ta-Na; Zhang, Xiao-Hong; Liu, Qi-Qing; Li, San-Qiang; Du, Qiu-Xiang

    2017-03-01

    The combined use of multiple markers is considered a promising strategy in estimating the age of wounds. We sought to develop an "up, no change, or down" system and to explore how to combine and use various parameters. In total, 78 Sprague Dawley rats were divided randomly into a control group and contusion groups of 4-, 8-, 12-, 16-, 20-, 24-, 28-, 32-, 36-, 40-, 44-, and 48-h post-injury (n=6 per group). A contusion was produced in the right limb of the rats under diethyl ether anesthesia by a drop-ball technique; the animals were sacrificed at certain time points thereafter, using a lethal dose of pentobarbital. Levels of PUM2, TAB2, GJC1, and CHRNA1 mRNAs were detected in contused muscle using real-time PCR. An up, no change, or down system was developed with the relative quantities of the four mRNAs recorded as black, dark gray, or light gray boxes, representing up-, no change, or down-regulation of the gene of interest during wound repair. The four transcripts were combined and used as a marker cluster for color model analysis of each contusion group. Levels of PUM2, TAB2, and GJC1 mRNAs decreased, whereas that of CHRNA1 increased in wound repair (P<0.05). The up, no change, or down system was adequate to distinguish most time groups with the color model. Thus, the proposed up, no change, or down system provide the means to determine the minimal periods of early wounds. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  16. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

    PubMed Central

    Taylor, Ethan Will; Ruzicka, Jan A.; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

  17. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry.

    PubMed

    Taylor, Ethan Will; Ruzicka, Jan A; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3' end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation.

  18. Deletion of intestinal epithelial insulin receptor attenuates high-fat diet-induced elevations in cholesterol and stem, enteroendocrine, and Paneth cell mRNAs.

    PubMed

    Andres, Sarah F; Santoro, M Agostina; Mah, Amanda T; Keku, J Adeola; Bortvedt, Amy E; Blue, R Eric; Lund, P Kay

    2015-01-15

    The insulin receptor (IR) regulates nutrient uptake and utilization in multiple organs, but its role in the intestinal epithelium is not defined. This study developed a mouse model with villin-Cre (VC) recombinase-mediated intestinal epithelial cell (IEC)-specific IR deletion (VC-IR(Δ/Δ)) and littermate controls with floxed, but intact, IR (IR(fl/fl)) to define in vivo roles of IEC-IR in mice fed chow or high-fat diet (HFD). We hypothesized that loss of IEC-IR would alter intestinal growth, biomarkers of intestinal epithelial stem cells (IESC) or other lineages, body weight, adiposity, and glucose or lipid handling. In lean, chow-fed mice, IEC-IR deletion did not affect body or fat mass, plasma glucose, or IEC proliferation. In chow-fed VC-IR(Δ/Δ) mice, mRNA levels of the Paneth cell marker lysozyme (Lyz) were decreased, but markers of other differentiated lineages were unchanged. During HFD-induced obesity, IR(fl/fl) and VC-IR(Δ/Δ) mice exhibited similar increases in body and fat mass, plasma insulin, mRNAs encoding several lipid-handling proteins, a decrease in Paneth cell number, and impaired glucose tolerance. In IR(fl/fl) mice, HFD-induced obesity increased circulating cholesterol; numbers of chromogranin A (CHGA)-positive enteroendocrine cells (EEC); and mRNAs encoding Chga, glucose-dependent insulinotrophic peptide (Gip), glucagon (Gcg), Lyz, IESC biomarkers, and the enterocyte cholesterol transporter Scarb1. All these effects were attenuated or lost in VC-IR(Δ/Δ) mice. These results demonstrate that IEC-IR is not required for normal growth of the intestinal epithelium in lean adult mice. However, our findings provide novel evidence that, during HFD-induced obesity, IEC-IR contributes to increases in EEC, plasma cholesterol, and increased expression of Scarb1 or IESC-, EEC-, and Paneth cell-derived mRNAs.

  19. Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors

    PubMed Central

    Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

    2016-01-01

    Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation—Pdx1, Neurogenin3, and MafA—efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2′-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2′-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy. PMID:27187823

  20. Deletion of intestinal epithelial insulin receptor attenuates high-fat diet-induced elevations in cholesterol and stem, enteroendocrine, and Paneth cell mRNAs

    PubMed Central

    Andres, Sarah F.; Santoro, M. Agostina; Mah, Amanda T.; Keku, J. Adeola; Bortvedt, Amy E.; Blue, R. Eric

    2014-01-01

    The insulin receptor (IR) regulates nutrient uptake and utilization in multiple organs, but its role in the intestinal epithelium is not defined. This study developed a mouse model with villin-Cre (VC) recombinase-mediated intestinal epithelial cell (IEC)-specific IR deletion (VC-IRΔ/Δ) and littermate controls with floxed, but intact, IR (IRfl/fl) to define in vivo roles of IEC-IR in mice fed chow or high-fat diet (HFD). We hypothesized that loss of IEC-IR would alter intestinal growth, biomarkers of intestinal epithelial stem cells (IESC) or other lineages, body weight, adiposity, and glucose or lipid handling. In lean, chow-fed mice, IEC-IR deletion did not affect body or fat mass, plasma glucose, or IEC proliferation. In chow-fed VC-IRΔ/Δ mice, mRNA levels of the Paneth cell marker lysozyme (Lyz) were decreased, but markers of other differentiated lineages were unchanged. During HFD-induced obesity, IRfl/fl and VC-IRΔ/Δ mice exhibited similar increases in body and fat mass, plasma insulin, mRNAs encoding several lipid-handling proteins, a decrease in Paneth cell number, and impaired glucose tolerance. In IRfl/fl mice, HFD-induced obesity increased circulating cholesterol; numbers of chromogranin A (CHGA)-positive enteroendocrine cells (EEC); and mRNAs encoding Chga, glucose-dependent insulinotrophic peptide (Gip), glucagon (Gcg), Lyz, IESC biomarkers, and the enterocyte cholesterol transporter Scarb1. All these effects were attenuated or lost in VC-IRΔ/Δ mice. These results demonstrate that IEC-IR is not required for normal growth of the intestinal epithelium in lean adult mice. However, our findings provide novel evidence that, during HFD-induced obesity, IEC-IR contributes to increases in EEC, plasma cholesterol, and increased expression of Scarb1 or IESC-, EEC-, and Paneth cell-derived mRNAs. PMID:25394660

  1. Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

    SciTech Connect

    Sonenberg, N.; Guertin, D.; Lee, K.A.W.

    1982-12-01

    Extracts form poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, the authors demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosiac virus 4 RNA, which is most probable devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

  2. Making scent of the presence and local translation of odorant receptor mRNAs in olfactory axons.

    PubMed

    Dubacq, Caroline; Fouquet, Coralie; Trembleau, Alain

    2014-03-01

    Rodents contain in their genome more than 1000 functional odorant receptor genes, which are specifically expressed by the olfactory sensory neurons projecting from the olfactory epithelium to the olfactory bulb. Strong evidence for the presence and local translation of odorant receptor mRNAs in the axon of olfactory sensory neurons was obtained, but no function has been assigned to these axonal mRNAs yet. The aim of this review is to discuss the evidence for the presence and local translation of odorant receptor mRNAs in olfactory sensory axons, and to speculate on their possible function in the wiring of the mouse olfactory sensory projections.

  3. In vivo translation and stability of trans-spliced mRNAs in nematode embryos.

    PubMed

    Cheng, Guofeng; Cohen, Leah; Mikhli, Claudette; Jankowska-Anyszka, Marzena; Stepinski, Janusz; Darzynkiewicz, Edward; Davis, Richard E

    2007-06-01

    Spliced leader trans-splicing adds a short exon, the spliced leader (SL), to pre-mRNAs to generate 5' ends of mRNAs. Addition of the SL in metazoa also adds a new cap to the mRNA, a trimethylguanosine (m(3)(2,2,7)GpppN) (TMG) that replaces the typical eukaryotic monomethylguanosine (m7GpppN)(m7G) cap. Both trans-spliced (m3(2,2,7)GpppN-SL-RNA) and not trans-spliced (m7GpppN-RNA) mRNAs are present in the same cells. Previous studies using cell-free systems to compare the overall translation of trans-spliced versus non-trans-spliced RNAs led to different conclusions. Here, we examine the contribution of m3(2,2,7)GpppG-cap and SL sequence and other RNA elements to in vivo mRNA translation and stability in nematode embryos. Although 70-90% of all nematode mRNAs have a TMG-cap, the TMG cap does not support translation as well as an m7G-cap. However, when the TMG cap and SL are present together, they synergistically interact and translation is enhanced, indicating both trans-spliced elements are necessary to promote efficient translation. The SL by itself does not act as a cap-independent enhancer of translation. The poly(A)-tail synergistically interacts with the mRNA cap enhancing translation and plays a greater role in facilitating translation of TMG-SL mRNAs. In general, recipient mRNA sequences between the SL and AUG and the 3' UTR do not significantly contribute to the translation of trans-spliced mRNAs. Overall, the combination of TMG cap and SL contribute to mRNA translation and stability in a manner typical of a eukaryotic m7G-cap and 5' UTRs, but they do not differentially enhance mRNA translation or stability compared to RNAs without the trans-spliced elements.

  4. Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

    PubMed

    Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

    1995-08-01

    The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

  5. ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression

    PubMed Central

    Yoshikawa, Takeshi; Wu, Jianfeng; Otsuka, Motoyuki; Kishikawa, Takahiro; Ohno, Motoko; Shibata, Chikako; Takata, Akemi; Han, Felicia; Kang, Young Jun; Chen, Chyi-Ying A.; Shyu, Ann-Bin; Han, Jiahuai; Koike, Kazuhiko

    2015-01-01

    The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function. PMID:26187994

  6. Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae.

    PubMed

    Bensidoun, Pierre; Raymond, Pascal; Oeffinger, Marlene; Zenklusen, Daniel

    2016-04-01

    Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we describe a detailed protocol for real time fluorescent RNA imaging using the PP7 bacteriophage coat protein, which allows mRNA detection with high spatial and temporal resolution in the yeast Saccharomyces cerevisiae, and can be applied to study various stages of mRNA metabolism. We describe the different parameters required for quantitative single molecule imaging in yeast, including strategies for genomic integration, expression of a PP7 coat protein GFP fusion protein, microscope setup and analysis strategies. We illustrate the method's use by analyzing the behavior of nuclear mRNA in yeast and the role of the nuclear basket in mRNA export.

  7. Putting an ‘End’ to HIV mRNAs: capping and polyadenylation as potential therapeutic targets

    PubMed Central

    2013-01-01

    Like most cellular mRNAs, the 5′ end of HIV mRNAs is capped and the 3′ end matured by the process of polyadenylation. There are, however, several rather unique and interesting aspects of these post-transcriptional processes on HIV transcripts. Capping of the highly structured 5′ end of HIV mRNAs is influenced by the viral TAT protein and a population of HIV mRNAs contains a trimethyl-G cap reminiscent of U snRNAs involved in splicing. HIV polyadenylation involves active repression of a promoter-proximal polyadenylation signal, auxiliary upstream regulatory elements and moonlighting polyadenylation factors that have additional impacts on HIV biology outside of the constraints of classical mRNA 3’ end formation. This review describes these post-transcriptional novelties of HIV gene expression as well as their implications in viral biology and as possible targets for therapeutic intervention. PMID:24330569

  8. Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development.

    PubMed

    Bao, Weier; Greenwold, Matthew J; Sawyer, Roger H

    2016-10-15

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and mRNAs

  9. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm.

    PubMed

    Vourekas, Anastassios; Alexiou, Panagiotis; Vrettos, Nicholas; Maragkakis, Manolis; Mourelatos, Zissimos

    2016-03-17

    The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) have a central role in genomic stability, which is inextricably linked to germ-cell formation, by forming Piwi ribonucleoproteins (piRNPs) that silence transposable elements. In Drosophila melanogaster and other animals, primordial germ-cell specification in the developing embryo is driven by maternal messenger RNAs and proteins that assemble into specialized messenger ribonucleoproteins (mRNPs) localized in the germ (pole) plasm at the posterior of the oocyte. Maternal piRNPs, especially those loaded on the Piwi protein Aubergine (Aub), are transmitted to the germ plasm to initiate transposon silencing in the offspring germ line. The transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process; mRNAs necessary for primordial germ-cell formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, the molecular identity of which remains unknown. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm, and interactions between Aub and Tudor are essential for the formation of germ granules. Here we show that Aub-loaded piRNAs use partial base-pairing characteristics of Argonaute RNPs to bind mRNAs randomly in Drosophila, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Notably, germ plasm mRNAs in drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus, complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. Our findings reveal an unexpected function for piRNP complexes in mRNA trapping that may be generally relevant to the function of animal germ granules.

  10. Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs.

    PubMed

    Yin, Zhikang; Wilson, Séan; Hauser, Nicole C; Tournu, Helene; Hoheisel, Jörg D; Brown, Alistair J P

    2003-05-01

    Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.

  11. Human selenoprotein P and S variant mRNAs with different numbers of SECIS elements and inferences from mutant mice of the roles of multiple SECIS elements.

    PubMed

    Wu, Sen; Mariotti, Marco; Santesmasses, Didac; Hill, Kristina E; Baclaocos, Janinah; Aparicio-Prat, Estel; Li, Shuping; Mackrill, John; Wu, Yuanyuan; Howard, Michael T; Capecchi, Mario; Guigó, Roderic; Burk, Raymond F; Atkins, John F

    2016-11-01

    Dynamic redefinition of the 10 UGAs in human and mouse selenoprotein P (Sepp1) mRNAs to specify selenocysteine instead of termination involves two 3' UTR structural elements (SECIS) and is regulated by selenium availability. In addition to the previously known human Sepp1 mRNA poly(A) addition site just 3' of SECIS 2, two further sites were identified with one resulting in 10-25% of the mRNA lacking SECIS 2. To address function, mutant mice were generated with either SECIS 1 or SECIS 2 deleted or with the first UGA substituted with a serine codon. They were fed on either high or selenium-deficient diets. The mutants had very different effects on the proportions of shorter and longer product Sepp1 protein isoforms isolated from plasma, and on viability. Spatially and functionally distinctive effects of the two SECIS elements on UGA decoding were inferred. We also bioinformatically identify two selenoprotein S mRNAs with different 5' sequences predicted to yield products with different N-termini. These results provide insights into SECIS function and mRNA processing in selenoprotein isoform diversity.

  12. Co-expression changes of lncRNAs and mRNAs in the cervical sympathetic ganglia in diabetic cardiac autonomic neuropathic rats.

    PubMed

    Li, Guilin; Sheng, Xuan; Xu, Yurong; Jiang, Huaide; Zheng, Chaoran; Guo, Jingjing; Sun, Shanshan; Yi, Zhihua; Qin, Shulan; Liu, Shuangmei; Gao, Yun; Zhang, Chunping; Xu, Hong; Wu, Bing; Zou, Lifang; Liang, Shangdong; Zhu, Gaochun

    2016-12-19

    Cardiac autonomic neuropathy in Type 2 diabetes (T2D) is often a devastating complication. Long non-coding RNAs (lncRNAs) have important effects on both normal development and disease pathogenesis. In this study, we explored the expression profiles of some lncRNAs involved in inflammation which may be co-expressed with messenger RNA (mRNA) in superior cervical and stellate ganglia after type 2 diabetic injuries. Total RNA isolated from 10 pairs of superior cervical and stellate ganglia in diabetic and normal male rats was hybridized to lncRNA arrays for detections. Pathway analysis indicated that the most significant gene ontology (GO) processes that were upregulated in diabetes were associated with immune response, cell migration, defense response, taxis, and chemotaxis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed that most of the target genes of the lncRNAs were located in cytokine-cytokine receptor interactions, the chemokine signaling pathway and cell adhesion molecules, which were involved in T2D. Gene co-expression network construction showed that the co-expression network in the experimental rats consisted of 268 regulation edges among 105 lncRNAs and 11 mRNAs. Our studies demonstrated the co-expression profile of lncRNAs and mRNAs in diabetic cardiac autonomic ganglia, suggesting possible roles for multiple lncRNAs as potential targets for the development of therapeutic strategies or biomarkers for diabetic cardiac autonomic neuropathy. © 2016 Wiley Periodicals, Inc.

  13. Human selenoprotein P and S variant mRNAs with different numbers of SECIS elements and inferences from mutant mice of the roles of multiple SECIS elements

    PubMed Central

    Wu, Sen; Mariotti, Marco; Santesmasses, Didac; Hill, Kristina E.; Baclaocos, Janinah; Aparicio-Prat, Estel; Li, Shuping; Mackrill, John; Wu, Yuanyuan; Howard, Michael T.; Capecchi, Mario; Guigó, Roderic; Burk, Raymond F.

    2016-01-01

    Dynamic redefinition of the 10 UGAs in human and mouse selenoprotein P (Sepp1) mRNAs to specify selenocysteine instead of termination involves two 3′ UTR structural elements (SECIS) and is regulated by selenium availability. In addition to the previously known human Sepp1 mRNA poly(A) addition site just 3′ of SECIS 2, two further sites were identified with one resulting in 10–25% of the mRNA lacking SECIS 2. To address function, mutant mice were generated with either SECIS 1 or SECIS 2 deleted or with the first UGA substituted with a serine codon. They were fed on either high or selenium-deficient diets. The mutants had very different effects on the proportions of shorter and longer product Sepp1 protein isoforms isolated from plasma, and on viability. Spatially and functionally distinctive effects of the two SECIS elements on UGA decoding were inferred. We also bioinformatically identify two selenoprotein S mRNAs with different 5′ sequences predicted to yield products with different N-termini. These results provide insights into SECIS function and mRNA processing in selenoprotein isoform diversity. PMID:27881738

  14. TOP mRNAs are translationally inhibited by a titratable repressor in both wheat germ extract and reticulocyte lysate.

    PubMed

    Biberman, Y; Meyuhas, O

    1999-08-13

    Vertebrate TOP mRNAs contain a 5' terminal oligopyrimidine tract (5' TOP), which is subject to selective translational repression in non-growing cells or in cell-free translation systems. In the present study, we monitored in vitro the effect of increasing amounts of a 16 nucleotides long oligoribonucleotide representing the 5' terminus of mouse ribosomal protein S16 mRNA on the translation of TOP and non-TOP mRNAs. Our results demonstrate that the wild-type sequence (but not its mutant counterparts) derepresses the translation of mRNAs containing 5' TOP motifs, but failed to stimulate the translation of non-TOP mRNAs, even if the latter differed only by a single nucleotide from their 5' TOP-containing counterparts. Similar results have been obtained with both wheat germ extract and rabbit reticulocyte lysate. It appears, therefore, that translational repression of TOP mRNAs is achieved in vitro by the accumulation of a titratable repressor rather than by the loss of an activator and that this repressor recognizes multiple TOP mRNAs with a diverse set of 5' TOP motifs.

  15. Polycistronic mRNAs code for polypeptides of the Vibrio harveyi luminescence system

    SciTech Connect

    Miyamoto, C.M.; Graham, A.D.; Boylan, M.; Evans, J.F.; Hasel, K.W.; Meighen, E.A.; Graham, A.F.

    1985-03-01

    DNA coding for the ..cap alpha.. and ..beta.. subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the ..cap alpha.. subunit as a hybridization probe, the authors identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the ..cap alpha.. and ..beta.. subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to the released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase ..cap alpha.. and ..beta.. subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.

  16. Novel factor rescues ribosomes trapped on non-stop mRNAs.

    PubMed

    Himeno, Hyouta

    2010-11-01

    Ribosomes are trapped at the 3′ ends of mRNAs that lack a natural stop codon. In bacteria, a reaction called trans-translation recycles ribosomes entrapped at such ‘non-stop’ mRNAs. The main player in trans-translation is tmRNA (SsrA-RNA), a bi-functional RNA that acts as both a tRNA and an mRNA. In the trans-translation reaction, alanine-charged tmRNA loads at the ribosomal A-site and translation shifts to the resume codon in tmRNA. Translation of tmRNA stops at a natural stop codon at the end of the small reading frame of tmRNA. In this way, the reaction simultaneously adds a peptide tag to the end of the nascent, incomplete polypeptide and recycles the stalled ribosomes. The peptide tag is recognized by cellular proteases that rapidly degrade the incomplete, potentially harmful polypeptides. The trans-translation reaction is not essential in most bacteria, raising the possibility that ribosomes stalled at non-stop mRNAs can be rescued by alternative routes. In this issue of Molecular Microbiology, Chadani et al. show that a novel translation factor, ArfA, can recycle a ribosome trapped at the 3′ end of a non-stop mRNA in the absence of trans-translation. AfrA is essential in the absence of tmRNA, showing that the two systems work in parallel to resolve stalled ribosomes.

  17. Regional Localization of Suspensor mRNAs during Early Embryo Development

    PubMed Central

    Weterings, Koen; Apuya, Nestor R.; Bi, Yuping; Fischer, Robert L.; Harada, John J.; Goldberg, Robert B.

    2001-01-01

    We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/β-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote. PMID:11701878

  18. Quantification and distribution of α1-adrenoceptor subtype mRNAs in human proximal urethra

    PubMed Central

    Nasu, Kimio; Moriyama, Nobuo; Fukasawa, Ritsu; Tsujimoto, Gozoh; Tanaka, Teruo; Yano, Junichi; Kawabe, Kazuki

    1998-01-01

    We performed RNase protection assays and in situ hybridization to investigate the ratio of the three α1-adrenoceptor subtype mRNAs, α1a, α1b and α1d, in human proximal urethra, and their localization in urethral cross-sections. As revealed by the RNase protection assays, α1a was the predominant subtype mRNA in both male and female urethral samples. α1d mRNA was detected only in the female sample, and α1b mRNA was not detected in any of the samples tested. The ratio of the abundance of the subtype mRNAs, α1a : α1b : α1d, was 100 : 0 : 0 in the male urethra and 90 : 0 : 10 in the female urethra.In situ hybridization studies showed no significant differences in the cross-sectional distribution of α1-adrenoceptor subtype mRNAs between male and female urethras. Intense α1a staining was observed in the smooth muscle of the urethra, but α1b and α1d staining was much less intense.Of the three cloned α1 subtypes, α1a is the most likely to be responsible for the contraction of the human urethra. Owing to the side effects of nonselective α1 drugs, α1-selective drugs may be clinically superior to nonselective drugs for the treatment of urethral disorders. PMID:9579721

  19. Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

    SciTech Connect

    Zhdanov, V. P.

    2010-10-15

    Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

  20. Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures.

    PubMed

    Leboy, P S; Beresford, J N; Devlin, C; Owen, M E

    1991-03-01

    We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the

  1. Complex processing patterns of mRNAs of the large ATP synthase operon in Arabidopsis chloroplasts.

    PubMed

    Malik Ghulam, Mustafa; Ghulam, Mustafa Malik; Courtois, Florence; Lerbs-Mache, Silva; Merendino, Livia

    2013-01-01

    Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5' and 3' end-definition. The current model for processing proposes that the 3' end of the upstream cistron transcripts and the 5' end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site. We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3' end, thus representing degradation products. We observe, instead, that the 5' ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3' ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have evolved multiple

  2. Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts

    PubMed Central

    Ghulam, Mustafa Malik; Courtois, Florence; Lerbs-Mache, Silva; Merendino, Livia

    2013-01-01

    Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5′ and 3′ end-definition. The current model for processing proposes that the 3′ end of the upstream cistron transcripts and the 5′ end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site. We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3′ end, thus representing degradation products. We observe, instead, that the 5′ ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3′ ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have

  3. Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation

    PubMed Central

    Zhao, Dongyan; Hamilton, John P.; Hardigan, Michael; Yin, Dongmei; He, Tao; Vaillancourt, Brieanne; Reynoso, Mauricio; Pauluzzi, Germain; Funkhouser, Scott; Cui, Yuehua; Bailey-Serres, Julia; Jiang, Jiming; Buell, C. Robin; Jiang, Ning

    2016-01-01

    Gene expression is controlled at transcriptional and post-transcriptional levels including decoding of messenger RNA (mRNA) into polypeptides via ribosome-mediated translation. Translational regulation has been intensively studied in the model dicot plant Arabidopsis thaliana, and in this study, we assessed the translational status [proportion of steady-state mRNA associated with ribosomes] of mRNAs by Translating Ribosome Affinity Purification followed by mRNA-sequencing (TRAP-seq) in rice (Oryza sativa), a model monocot plant and the most important food crop. A survey of three tissues found that most transcribed rice genes are translated whereas few transposable elements are associated with ribosomes. Genes with short and GC-rich coding regions are overrepresented in ribosome-associated mRNAs, suggesting that the GC-richness characteristic of coding sequences in grasses may be an adaptation that favors efficient translation. Transcripts with retained introns and extended 5′ untranslated regions are underrepresented on ribosomes, and rice genes belonging to different evolutionary lineages exhibited differential enrichment on the ribosomes that was associated with GC content. Genes involved in photosynthesis and stress responses are preferentially associated with ribosomes, whereas genes in epigenetic regulation pathways are the least enriched on ribosomes. Such variation is more dramatic in rice than that in Arabidopsis and is correlated with the wide variation of GC content of transcripts in rice. Taken together, variation in the translation status of individual transcripts reflects important mechanisms of gene regulation, which may have a role in evolution and diversification. PMID:27852012

  4. Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences

    PubMed Central

    Boehm, Volker; Gerbracht, Jennifer V.; Marx, Marie-Charlotte; Gehring, Niels H.

    2016-01-01

    The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5′–3′ mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3′ UTRs of cytokine mRNAs reveals the contribution of 5′–3′ decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses. PMID:27917860

  5. Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap.

    PubMed

    Gaupels, Frank; Buhtz, Anja; Knauer, Torsten; Deshmukh, Sachin; Waller, Frank; van Bel, Aart J E; Kogel, Karl-Heinz; Kehr, Julia

    2008-01-01

    Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 mul of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.

  6. Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function.

    PubMed

    Osman, Abdimajid; Hitzler, Walter E; Meyer, Claudius U; Landry, Patricia; Corduan, Aurélie; Laffont, Benoit; Boilard, Eric; Hellstern, Peter; Vamvakas, Eleftherios C; Provost, Patrick

    2015-01-01

    Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen+ ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin+ UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.

  7. Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function

    PubMed Central

    Osman, Abdimajid; Hitzler, Walter E.; Meyer, Claudius U.; Landry, Patricia; Corduan, Aurélie; Laffont, Benoit; Boilard, Eric; Hellstern, Peter; Vamvakas, Eleftherios C.

    2015-01-01

    Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen + ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin + UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established. PMID:24749844

  8. YTHDC1 Mediates Nuclear Export of N(6)-methyladenosine Methylated mRNAs.

    PubMed

    Roundtree, Ian A; Luo, Guan-Zheng; Zhang, Zijie; Wang, Xiao; Zhou, Tao; Cui, Yiquang; Sha, Jiahao; Huang, Xingxu; Guerrero, Laura; Xie, Phil; He, Emily; Shen, Bin; He, Chuan

    2017-10-06

    N(6)-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA), and plays critical roles in RNA biology. The function of this modification is mediated by m(6)A-selective 'reader' proteins of the YTH family, which incorporate m(6)A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m(6)A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m(6)A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m(6)A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

  9. Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes.

    PubMed

    Xu, Shaohang; Zhou, Ruo; Ren, Zhe; Zhou, Baojin; Lin, Zhilong; Hou, Guixue; Deng, Yamei; Zi, Jin; Lin, Liang; Wang, Quanhui; Liu, Xin; Xu, Xun; Wen, Bo; Liu, Siqi

    2015-12-04

    Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins. Total of 1501 missing proteins were found by neither RNC-mRNA nor MS/MS in the three liver cancer cell lines. For these missing proteins, some are expected higher hydrophobicity, unsuitable detection, or sensory functions as properties at the protein level, while some are predicted to have nonexpressing chromatin structures on the corresponding gene level. With further integrated analysis, we could attribute 93% of them (1391/1501) to these causal factors, which result in the expression products scarcely detected by RNA-seq or MS/MS.

  10. Operon mRNAs are organized into ORF-centric structures that predict translation efficiency

    PubMed Central

    Burkhardt, David H; Rouskin, Silvi; Zhang, Yan; Li, Gene-Wei; Weissman, Jonathan S; Gross, Carol A

    2017-01-01

    Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF. DOI: http://dx.doi.org/10.7554/eLife.22037.001 PMID:28139975

  11. Identification, Mapping, and In Vitro Translation of Campoletis sonorensis Virus mRNAs from Parasitized Heliothis virescens Larvae

    PubMed Central

    Blissard, Gary W.; Vinson, S. Bradleigh; Summers, Max D.

    1986-01-01

    Expression of Campoletis sonorensis virus (CsV) in parasitized Heliothis virescens larvae was investigated by Northern blot analysis of poly(A)+ mRNAs isolated from H. virescens larvae at various times after parasitization by C. sonorensis. At least 12 CsV mRNAs were detected in parasitized H. virescens larvae. Injection of nonparasitized H. virescens larvae with purified CsV resulted in a pattern of viral mRNAs similar to that observed in naturally parasitized larvae. With CsV DNA restriction fragments which contained expressed sequences, individual CsV mRNAs were mapped to the superhelical DNAs of the viral genome. Two gene-specific probes, which consisted of cloned S1 nuclease-protected restriction fragments, each hybridized to several CsV superhelical DNAs, suggesting that some CsV genes may be shared on several superhelical DNAs. Cloned restriction fragments containing sequences which flank the expressed sequences also hybridized to numerous CsV superhelical DNAs. Some CsV proteins were identified by in vitro translation of hybrid-selected CsV mRNAs. Images PMID:16789254

  12. Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

    PubMed Central

    Ote, Isabelle; Lebrun, Marielle; Vandevenne, Patricia; Bontems, Sébastien; Medina-Palazon, Cahora; Manet, Evelyne; Piette, Jacques; Sadzot-Delvaux, Catherine

    2009-01-01

    Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export. PMID:19924249

  13. Varicella-zoster virus IE4 protein interacts with SR proteins and exports mRNAs through the TAP/NXF1 pathway.

    PubMed

    Ote, Isabelle; Lebrun, Marielle; Vandevenne, Patricia; Bontems, Sébastien; Medina-Palazon, Cahora; Manet, Evelyne; Piette, Jacques; Sadzot-Delvaux, Catherine

    2009-11-18

    Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

  14. Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts.

    PubMed Central

    Varga, J; Rosenbloom, J; Jimenez, S A

    1987-01-01

    It has been previously shown that transforming growth factor beta (TGF beta) is capable of stimulating fibroblast collagen and fibronectin biosynthesis. The purpose of this study was to examine the mechanisms involved in TGF beta stimulation of fibroblast biosynthetic activity. Our results indicate that TGF beta causes a marked enhancement of the production of types I and III collagens and fibronectin by cultured normal human dermal fibroblasts. The rate of collagen production by fibroblasts exposed to TGF beta was 2-3-fold greater than that of control cells. These effects were associated with a 2-3-fold increase in the steady-state amounts of types I and III collagen mRNAs and a 5-8-fold increase in the amounts of fibronectin mRNAs as determined by dot-blot hybridization with specific cloned cDNA probes. In addition, the increased production of collagen and fibronectin and the increased amounts of their corresponding mRNAs remained elevated for at least 72 h after removal of TGF beta. These findings suggest that TGF beta may play a major role in the normal regulation of extracellular matrix production in vivo and may contribute to the development of pathological states of fibrosis. Images Fig. 1. Fig. 4. PMID:3501287

  15. An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains

    PubMed Central

    Jäger, Dominik; Pernitzsch, Sandy R.; Richter, Andreas S.; Backofen, Rolf; Sharma, Cynthia M.; Schmitz, Ruth A.

    2012-01-01

    We report on the characterization and target analysis of the small (s)RNA162 in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5′ fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA162 (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA162 is crucial for target interactions. Furthermore, our results indicate that sRNA162-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 5′ end of sRNA162 targets the 5′-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA162 acts as an antisense (as)RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated. PMID:22965121

  16. Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites.

    PubMed

    Guo, Junli; Zheng, Wenyu; Wang, Yuehua; Li, Yao; Lu, Siqi; Feng, Xianmin

    2014-02-14

    A strategy of the parasitic protozoan Giardia lamblia to evade attack from the host immune system is periodic changes of its surface antigen, a member of the variant surface protein (VSP) family. A post-transcriptional gene silencing mechanism has been proposed to explain the presence of only one among many possible VSPs at any time. To investigate this phenomenon further, we extracted total RNA from cultured trophozoites of the G. lamblia C2 isolate, and cDNA was reverse-transcribed from the RNA. Sense and anti-sense VSPs were amplified from the total cDNA using nested PCR with primers designed from the 3'-conserved region and the known 5' or 3' end of the cDNA library. Sequence analyses of the amplified products revealed more than 34 full-length antisense VSPs and a smear of sense VSPs. Sequence alignments and comparisons revealed that these VSPs contained variable N-termini and conserved C-termini, and could be classified into 5 clades based on the sizes and variations of the N-terminal sequence. All antisense VSPs existed in the sense forms, but no corresponding antisense VSP existed for sense RNA (snsRNA) 16. The coexistence of sense and antisense VSP mRNAs in cultured G. lamblia supports the post-transcriptional regulation of VSP expression. We propose that VSPs transcribed simultaneously in the sense and antisense forms form double-stranded RNAs (dsRNAs) which are degraded by the Dicer endonuclease, while a VSP without an antisense transcription (e.g., snsRNA16) will be expressed on the surface of Giardia. In addition, in the course of this investigation VSPs were identified that were previously not known. PCR-based amplification of specific sense and antisense VSP cDNAs can be used to identify the specific VSP on G. lamblia trophozoites, which is easier than using specific monoclonal antibody approaches. Copyright © 2014. Published by Elsevier Inc.

  17. The specific expression pattern of globin mRNAs in Tibetan chicken during late embryonic stage under hypoxia.

    PubMed

    Liu, C; Zhang, L F; Li, N

    2013-04-01

    Tibetan chicken (Gallus gallus) is a specific chicken breed with strong ability to resist hypoxia, especially during embryonic stage. Though this breed has lived in Tibet plateau for thousands of years, the adaptive mechanism in response to hypoxia is still unknown. In order to obtain a better understanding of the mechanism of hypoxic adaptability in high altitude, we analyzed the mRNA expression pattern of globins in the present study. The fertilized eggs from Tibetan chicken and dwarf recessive white chicken breeds were incubated under normoxic (21% O2) and hypoxic (13% O2) conditions, equivalent to the altitude of 3600m. We observed that Tibetan chicken embryos had higher hatchability (48%) in hypoxia than their lowland controls (7.8%). Northern blot showed that globin mRNA expression in Tibetan chicken embryos differed greatly from lowland controls under hypoxia. The expressions of four dominant globin mRNAs, named α(A), α(D), β(A) and β(H), were significantly induced under hypoxia. Tibetan chicken embryos had lower globin mRNA level in red cells than that of lowland controls at day 19 (P<0.05). Based on real-time PCR the same result was confirmed. Furthermore, we observed accumulation of globins induced by hypoxia in red cells by performing the separation of globin analysis, showing higher level of globins in red cells of Tibetan chicken embryos than that of lowland chicken embryos. Overall, our results provide new evidence that flexible regulation of globins at the level of transcription and translation may play a role in allowing the Tibetan chicken embryo to resist hypoxia. Copyright © 2012. Published by Elsevier Inc.

  18. Administration of haloperidol with biperiden reduces mRNAs related to the ubiquitin-proteasome system in mice.

    PubMed

    Iwata, Shin-Ichi; Morioka, Hirofumi; Iwabuchi, Mika; Shinohara, Kazuya; Maeda, Maki; Shimizu, Takao; Miyata, Atsuro

    2005-06-15

    In order to find molecules affected by administration of an antipsychotic drug with an antimuscarinic drug, which is a common prescription used to prevent extrapyramidal adverse effects caused by the antipsychotic drugs, gene expression profiling in the frontal cortex was studied in mice. After 14 days of administration with 2 mg/kg haloperidol, a typical antipsychotic drug, and 2 mg/kg biperiden, a high-affinity antagonist for muscarinic receptors in the brain, approximately 500 mRNAs related to synaptic function were investigated. The levels of the mRNAs related to the ubiquitin-related systems were significantly reduced after the combined administration. However, the separate administration of either haloperidol or biperiden had little effect on the levels of the mRNAs. This result suggests that coadministration of haloperidol and biperiden specifically affects the ubiquitin-related system.

  19. miR-190 is upregulated in Epstein-Barr Virus type I latency and modulates cellular mRNAs involved in cell survival and viral reactivation.

    PubMed

    Cramer, Elizabeth M; Shao, Ying; Wang, Yan; Yuan, Yan

    2014-09-01

    Epstein-Barr Virus (EBV) is a prevalent human pathogen infecting over 90% of the population. Much of the success of the virus is attributed to its ability to maintain latency. The detailed mechanisms underlying the establishment and maintenance of EBV latency remain poorly understood. A microRNA profiling study revealed differential expression of many cellular miRNAs between types I and III latency cells, suggesting cellular miRNAs may play roles in regulating EBV latency. mir-190 is the most differentially up-regulated miRNA in type I latency cells as compared with type III latency cells and the up-regulation appears to be attributed to EBER RNAs that express in higher levels in type I latency cells than type III cells. With the aide of a lentiviral overexpression system and microarray analysis, several cellular mRNAs are identified as potential targets of mir-190. By targeting TP53INP1, miR-190 enhances cell survival by preventing apoptosis and relieving G0/G1 cell cycle arrest. Additionally, miR-190 down-regulates NR4A3, a cellular immediate-early gene for EBV reactivation, and inhibits the expression of the viral immediate-early gene bzlf1 and viral lytic DNA replication. Taken together, our data revealed a mechanism that EBV utilizes a cellular microRNA to promote host cell survival and prevent virus from entering lytic life cycle for latency maintenance.

  20. A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues

    PubMed Central

    Lyons, Shawn M.; Cunningham, Clark H.; Welch, Joshua D.; Groh, Beezly; Guo, Andrew Y.; Wei, Bruce; Whitfield, Michael L.; Xiong, Yue; Marzluff, William F.

    2016-01-01

    Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins. PMID:27402160

  1. Translation elongation can control translation initiation on eukaryotic mRNAs

    PubMed Central

    Chu, Dominique; Kazana, Eleanna; Bellanger, Noémie; Singh, Tarun; Tuite, Mick F; von der Haar, Tobias

    2014-01-01

    Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability. PMID:24357599

  2. Analyses of gene function in amphioxus embryos by microinjection of mRNAs and morpholino oligonucleotides.

    PubMed

    Holland, Linda Z; Onai, Takayuki

    2011-01-01

    The invertebrate chordate amphioxus (Branchiostoma), which is the most basal living chordate, has become an accepted model for the vertebrate ancestor in studies of development and evolution. Amphioxus resembles vertebrates in regard to morphology, developmental gene expression, and gene function. In addition, the amphioxus genome has representatives of most vertebrate gene families. Although it has not undergone the two rounds of whole genome duplications that occurred early in the vertebrate lineage, the amphioxus genome has retained considerable synteny with vertebrate genomes. Thus, studies of genes and development in amphioxus embryos can reveal the fundamental genetic basis of the vertebrate body plan, giving insights into the developmental mechanisms of such organs as the somites, pharynx, kidney, and the central nervous system. Moreover, amphioxus is very useful for understanding how these characters evolved. This chapter details methods for microinjection of amphioxus eggs with mRNAs or morpholino antisense oligonucleotides to analyze gene networks operating in early development.

  3. mRNAs encoding aquaporins are present during murine preimplantation development.

    PubMed

    Offenberg, H; Barcroft, L C; Caveney, A; Viuff, D; Thomsen, P D; Watson, A J

    2000-12-01

    The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation.

  4. Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

    PubMed Central

    Gaupels, Frank; Buhtz, Anja; Knauer, Torsten; Deshmukh, Sachin; Waller, Frank; van Bel, Aart J. E.; Kogel, Karl-Heinz; Kehr, Julia

    2008-01-01

    Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies. PMID:18632729

  5. Neuronal Expression of CB2 Cannabinoid Receptor mRNAs in the Mouse Hippocampus

    PubMed Central

    Li, Yong; Kim, Jimok

    2015-01-01

    In the brain, CB1 cannabinoid receptors primarily mediate the effects of cannabinoids, but CB2 cannabinoid receptors (CB2Rs) have recently been discovered in the nervous system and also implicated in neuromodulatory roles. To understand the mechanisms of CB2R functions in the brain, it is essential to localize CB2Rs, but the types of cells expressing CB2Rs have been controversial. Unequivocal localization of CB2Rs in the brain has been impeded in part by the low expression levels of CB2Rs and poor specificity of detection methods. Here, we used an ultrasensitive and specific in situ hybridization method called the RNAscope to determine the spatial pattern of CB2R mRNA expression in the mouse hippocampus. CB2R mRNAs were mostly expressed in a subset of excitatory and inhibitory neurons in the CA1, CA3 and dentate gyrus areas, but rarely in microglia. CB2R knock-out mice were used as a negative control. Using the quantitative real-time polymerase chain reaction, we also found that the temporal pattern of CB2R mRNA expression was stable during postnatal development. Consistent with previous reports, the immunological detection of CB2Rs was not reliable, implying extremely low levels of the protein expression and/or insufficient specificity of the current anti-CB2R antibodies. Our findings of the expression patterns of CB2R mRNAs may help determine the cell types involved in, and hence the mechanisms of, the CB2R-mediated neuromodulation. PMID:26515747

  6. Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p

    PubMed Central

    Saavedra, Claudio A.; Hammell, Christopher M.; Heath, Catherine V.; Cole, Charles N.

    1997-01-01

    We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)+ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)+ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways. PMID:9353254

  7. Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos

    PubMed Central

    1994-01-01

    A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence indicate that the interaction of microtubules with ribosomes is specific: a distinct stalk-like structure appears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribosomes in these preparations depends on the presence of intact microtubules. Five specific mRNAs are enriched with the microtubule- bound ribosomes, indicating that translation of specific proteins may occur on the microtubule scaffolding in vivo. PMID:7962079

  8. Systematic analysis of berberine-induced signaling pathway between miRNA clusters and mRNAs and identification of mir-99a ∼ 125b cluster function by seed-targeting inhibitors in multiple myeloma cells.

    PubMed

    Feng, Maoxiao; Luo, Xiaochuang; Gu, Chunming; Li, Yumin; Zhu, Xuejiao; Fei, Jia

    2015-01-01

    Berberine (BBR) is a natural alkaloid derived from a traditional Chinese herbal medicine. However, the exact mechanisms underlying the different effects of berberine on MM cells have not been fully elucidated. A systematic analysis assay integrated common signaling pathways modulated by the 3 miRNA clusters and mRNAs in MM cells after BBR treatment. The role of the mir-99a ∼ 125b cluster, an important oncomir in MM, was identified by comparing the effects of t-anti-mirs with complete complementary antisense locked nucleic acids (LNAs) against mature mir-125b (anti-mir-125b). Three miRNAs clusters (miR-99a ∼ 125b, miR-17 ∼ 92 and miR-106 ∼ 25) were significantly down-regulated in BBR-treated MM cells and are involved in multiple cancer-related signaling pathways. Furthermore, the top 5 differentially regulated genes, RAC1, NFκB1, MYC, JUN and CCND1 might play key roles in the progression of MM. Systematic integration revealed that 3 common signaling pathways (TP53, Erb and MAPK) link the 3 miRNA clusters and the 5 key mRNAs. Meanwhile, both BBR and seed-targeting t-anti-mir-99a ∼ 125b cluster LNAs significantly induced apoptosis, G2-phase cell cycle arrest and colony inhibition. our results suggest that BBR suppresses multiple myeloma cells, partly by down-regulating the 3 miRNA clusters and many mRNAs, possibly through TP53, Erb and MAPK signaling pathways. The mir-99a ∼ 125b cluster might be a novel target for MM treatment. These findings provide new mechanistic insight into the anticancer effects of certain traditional Chinese herbal medicine compounds.

  9. Characterization of target mRNAs for grapevine microRNAs with an integrated strategy of modified RLM-RACE, newly developed PPM-RACE and qPCRs.

    PubMed

    Wang, Chen; Han, Jian; Korir, Nicholas Kibet; Wang, Xicheng; Liu, Hong; Li, Xiaoying; Leng, Xiangpeng; Fang, Jinggui

    2013-07-01

    MicroRNAs (miRNAs) regulate target gene expression by mediating target gene cleavage or inhibition of translation at transcriptional and post-transcriptional levels in higher plants. Until now, many grapevine microRNAs (Vv-miRNAs) have been identified and quite a number of miRNA target genes were also verified by various analysis. However, global interaction of miRNAs with their target genes still remained to perform more research. We reported experimental validation of a number of miRNA target genes in table grapevine that had been previously identified by bioinformatics in our earlier studies. To verify more predicted target genes of Vv-miRNAs and elucidate the modes by which these Vv-miRNAs work on their target genes, 31 unverified potential target genes for 18 Vv-miRNAs were experimentally verified by a new integrated strategy employing a modified 5'-RLM-RACE (RNA ligase-mediated 5' rapid amplification of cDNA ends), 3'-PPM-RACE (poly(A) polymerase-mediated 3' rapid amplification of cDNA ends) and qRT-PCRs of cleavage products. The results showed that these Vv-miRNAs negatively regulated expression of their target messenger RNAs (mRNAs) through guiding corresponding target mRNA cleavage, of which about 94.4% Vv-miRNAs cleaved their target mRNAs mainly at the tenth nucleotide of 5'-end of miRNAs. Expression levels of both miRNAs and their target mRNAs in eight tissues exhibited inverse relationships, and expressions both of cleaved targets and miRNAs indicated a cleavage mode of Vv-miRNAs on their target genes. Our results confirm the importance of Vv-miRNAs in grapevine growth and development, and suggest more study on Vv-miRNAs and targets can enrich the knowledge of miRNA mediated-regulation in grapevine.

  10. Comparative expression profiles of mRNAs and microRNAs among human mesenchymal stem cells derived from breast, face, and abdominal adipose tissues.

    PubMed

    Wang, Kai-Hung; Kao, An-Pei; Singh, Sher; Yu, Sung-Liang; Kao, Li-Pin; Tsai, Zong Yun; Lin, Sin-Daw; Li, Steven Shoei-Lung

    2010-03-01

    We determined the expression of both mRNAs and microRNAs (miRNAs) from human mesenchymal stem cells BM19, FM30, and AM3, which is derived from breast, face, and abdominal adipose tissues, respectively. BM19, FM30, and AM3 cells exhibited considerably similar mRNA profiles, and their 1,038 abundantly common genes were involved in regulating six cell adhesion and three cytoskeleton remodeling processes among the top ten GeneGo canonical pathway maps. The 39 most abundant miRNAs in AM3 cells were expressed at very similar levels in BM19 cells. However, seven abundant miRNAs (miR-19b, miR-320, miR-186, miR-199a, miR-339, miR-99a, and miR-152) in AM3 cells were expressed at much lower levels than that in FM30 cells, and 38 genes targeted by these miRNAs were consequently upregulated more than 3-fold in FM30 cells compared with AM3 cells. Therefore, autologous abdominal adipose-derived mesenchymal stem cells are suitable for tissue engineering of breast reconstruction because of very similar expression profiles of mRNAs and miRNAs between AM3 and BM19 cells. Conversely, abdominal AM3 cells might not be suitable for facial rejuvenation, since the 38 highly expressed genes targeted by miRNAs in FM30 cells might play an important role(s) in the development of facial tissue.

  11. Destabilization of CARP mRNAs by aloe-emodin contributes to caspase-8-mediated p53-independent apoptosis of human carcinoma cells.

    PubMed

    Lin, Meng-Liang; Lu, Yao-Cheng; Su, Hong-Lin; Lin, Hsin-Ting; Lee, Chuan-Chun; Kang, Shang-En; Lai, Tan-Chen; Chung, Jing-Gung; Chen, Shih-Shun

    2011-04-01

    Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.

  12. Regional heterogeneity of expression of renal NPRs, TonEBP, and AQP-2 mRNAs in rats with acute kidney injury.

    PubMed

    Cha, Seung Ah; Park, Byung Mun; Jung, Yu Jin; Kim, Soo Mi; Kang, Kyung Pyo; Kim, Won; Kim, Suhn Hee

    2015-07-01

    To understand the pathophysiology of ischemia/reperfusion (I/R) - induced acute kidney injury (AKI), the present study defined changes in renal function, plasma renotropic hormones and its receptors in the kidney 2, 5, or 7 days after 45 min-renal ischemia in rats. Blood urea nitrogen, plasma creatinine, and osmolarity increased 2 days after I/R injury and tended to return to control level 7 days after I/R injury. Decreased renal function tended to return to control level 5 days after I/R injury. However, plasma concentrations of atrial natriuretic peptide and renin did not change. In control kidney, natriuretic peptide receptor (NPR)-A, -B and -C mRNAs were highly expressed in medulla (ME), inner cortex (IC), and outer cortex (OC), respectively, and tonicity-responsive enhancer binding protein (TonEBP), auqaporin-2 (AQP-2) and eNOS mRNAs were highly expressed in ME. NPR-A and -B mRNA expressions were markedly decreased 2 days after I/R injury. On 5 days after I/R injury, NPR-A mRNA expression increased in OC and recovered to control level in IC but not in ME. NPR-B mRNA expression was increased in OC, and recovered to control level in IC and ME. NPR-C mRNA expression was markedly decreased in OC 2 and 5 days after I/R injury. TonEBP, APQ-2 and eNOS mRNA expressions were markedly decreased 2 days after I/R injury and did not recover in ME 7 days after I/R injury. Therefore, we suggest that there is a regional heterogeneity of regulation of renal NPRs, TonEBP, and APQ-2 mRNA in AKI.

  13. Hormonal and photoperiodic modulation of testicular mRNAs coding for inhibin/activin subunits and follistatin in Clethrionomys glareolus, Schreber.

    PubMed

    Tähkä, K M; Kaipia, A; Toppari, J; Tähkä, S; Tuuri, T; Tuohimaa, P

    1998-07-01

    Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

  14. Two separate modules of the conserved regulatory RNA AbcR1 address multiple target mRNAs in and outside of the translation initiation region

    PubMed Central

    Overlöper, Aaron; Kraus, Alexander; Gurski, Rosemarie; Wright, Patrick R; Georg, Jens; Hess, Wolfgang R; Narberhaus, Franz

    2014-01-01

    The small RNA AbcR1 regulates the expression of ABC transporters in the plant pathogen Agrobacterium tumefaciens, the plant symbiont Sinorhizobium meliloti, and the human pathogen Brucella abortus. A combination of proteomic and bioinformatic approaches suggested dozens of AbcR1 targets in A. tumefaciens. Several of these newly discovered targets are involved in the uptake of amino acids, their derivatives, and sugars. Among the latter is the periplasmic sugar-binding protein ChvE, a component of the virulence signal transduction system. We examined 16 targets and their interaction with AbcR1 in close detail. In addition to the previously described mRNA interaction site of AbcR1 (M1), the CopraRNA program predicted a second functional module (M2) as target-binding site. Both M1 and M2 contain single-stranded anti-SD motifs. Using mutated AbcR1 variants, we systematically tested by band shift experiments, which sRNA region is responsible for mRNA binding and gene regulation. On the target site, we find that AbcR1 interacts with some mRNAs in the translation initiation region and with others far into their coding sequence. Our data show that AbcR1 is a versatile master regulator of nutrient uptake systems in A. tumefaciens and related bacteria. PMID:24921646

  15. Single-molecule imaging of {beta}-actin mRNAs in the cytoplasm of a living cell

    SciTech Connect

    Yamagishi, Mai; Ishihama, Yo; Shirasaki, Yoshitaka; Kurama, Hideki; Funatsu, Takashi

    2009-04-15

    {beta}-Actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most {beta}-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D{sub MACRO}) at the leading edge was 0.3 {mu}m{sup 2}/s. On the other hand, D{sub MACRO} in the perinuclear region was 0.02 {mu}m{sup 2}/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize {beta}-actin mRNAs, led to an increase in D{sub MACRO} to 0.2 {mu}m{sup 2}/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of {beta}-actin mRNA.

  16. SV40-IMMORTALIZED NON-TUMORIGENIC AND TUMORIGENIC CELL LINES DIFFER IN EXPRESSION OF HALLMARK VIRAL RESPONSE MRNAS

    EPA Science Inventory

    SV40-Immortalized Non-Tumorigenic and Tumorigenic Cell Lines Differ in Expression of Hallmark Viral Response mRNAs.

    Prior to the use of an in vitra/in viva transformation system to examine the tumorigenic activity of environmental contaminants, in vitra gene expression pa...

  17. Translation of nad9 mRNAs in mitochondria from Solanum tuberosum is restricted to completely edited transcripts.

    PubMed Central

    Grohmann, L; Thieck, O; Herz, U; Schröder, W; Brennicke, A

    1994-01-01

    The pool of partially and completely edited mRNAs present in plant mitochondria could potentially be translated into a mixture of divergent proteins. This possibility was investigated for the nad9 gene in potato by characterization of the mRNA population and the corresponding protein sequence. The deduced amino acid sequence of the nad9 gene product has significant similarity to the nuclear-encoded 30 kDa subunit of the bovine and Neurospora NADH:ubiquinone oxidoreductase (complex I) and to the chloroplast ndhJ gene product. Immunoprecipitation of a 27 kDa in-organello 35S labelled mitochondrial translation product with an antibody directed against the wheat nad9 gene product demonstrates its functional expression in potato and wheat. Comparison of the nad9 genomic DNA and cDNA sequences reveals seven codons to be changed by a C to U RNA-editing. Direct sequencing of RT-PCR products derived from cDNAs of different tissues of potato plants shows the presence of a significant portion of only partially edited nad9 transcripts in the various tissues. Amino acid sequencing of internal peptides of the isolated 27 kDa protein from potato tubers demonstrates homogenous translation products of only completely edited nad9 mRNAs even in the presence of partially edited mRNAs. This result suggests a pretranslational selection between edited and incompletely edited mRNAs in plant mitochondria. Images PMID:8078764

  18. Single-molecule imaging of beta-actin mRNAs in the cytoplasm of a living cell.

    PubMed

    Yamagishi, Mai; Ishihama, Yo; Shirasaki, Yoshitaka; Kurama, Hideki; Funatsu, Takashi

    2009-04-15

    Beta-actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most beta-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D(MACRO)) at the leading edge was 0.3 microm(2)/s. On the other hand, D(MACRO) in the perinuclear region was 0.02 microm(2)/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize beta-actin mRNAs, led to an increase in D(MACRO) to 0.2 microm(2)/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of beta-actin mRNA.

  19. Granules Harboring Translationally Active mRNAs Provide a Platform for P-Body Formation following Stress

    PubMed Central

    Lui, Jennifer; Castelli, Lydia M.; Pizzinga, Mariavittoria; Simpson, Clare E.; Hoyle, Nathaniel P.; Bailey, Kathryn L.; Campbell, Susan G.; Ashe, Mark P.

    2014-01-01

    Summary The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems. PMID:25437551

  20. Protein-tyrosine-kinase-dependent expression of cyclo-oxygenase-1 and -2 mRNAs in human endothelial cells.

    PubMed Central

    Hirai, K; Takayama, H; Tomo, K; Okuma, M

    1997-01-01

    Endothelial cells possess constitutive or inducible cyclo-oxygenase (COX) isoenzymes for prostacyclin production, but the mechanisms for their expression are largely unknown. We found that vanadate, an inhibitor of protein-tyrosine phosphatases, induced the expression of two COX isoenzyme mRNAs in human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner. Vanadate also stimulated an increase in COX-2 protein levels, but did not affect significantly the levels of constitutively expressed COX-1 protein. Synergistic enhancement of expression of the two COX isoenzyme mRNAs was observed on stimulation of HUVEC with vanadate plus interleukin-1alpha. Tyrphostin-47, which as an inhibitor of protein-tyrosine kinases abolished vanadate-induced protein-tyrosine phosphorylation, inhibited expression of the two COX isoenzyme mRNAs in HUVEC stimulated with vanadate or interleukin-1alpha. These data provide conclusive evidence that activation of protein-tyrosine kinases is causally linked to expression of the mRNAs for the two COX isoenzymes in HUVEC. PMID:9065752

  1. SV40-IMMORTALIZED NON-TUMORIGENIC AND TUMORIGENIC CELL LINES DIFFER IN EXPRESSION OF HALLMARK VIRAL RESPONSE MRNAS

    EPA Science Inventory

    SV40-Immortalized Non-Tumorigenic and Tumorigenic Cell Lines Differ in Expression of Hallmark Viral Response mRNAs.

    Prior to the use of an in vitra/in viva transformation system to examine the tumorigenic activity of environmental contaminants, in vitra gene expression pa...

  2. Multiple microRNAs within the 14q32 cluster target the mRNAs of major type 1 diabetes autoantigens IA-2, IA-2β, and GAD65.

    PubMed

    Abuhatzira, Liron; Xu, Huanyu; Tahhan, Georges; Boulougoura, Afroditi; Schäffer, Alejandro A; Notkins, Abner L

    2015-10-01

    Islet antigen (IA)-2, IA-2β, and glutamate decarboxylase (GAD65) are major autoantigens in type 1 diabetes (T1D). Autoantibodies to these autoantigens appear years before disease onset and are widely used as predictive markers. Little is known, however, about what regulates the expression of these autoantigens. The present experiments were initiated to test the hypothesis that microRNAs (miRNAs) can target and affect the levels of these autoantigens. Bioinformatics was used to identify miRNAs predicted to target the mRNAs coding IA-2, IA-2β, and GAD65. RNA interference for the miRNA processing enzyme Dicer1 and individual miRNA mimics and inhibitors were used to confirm the effect in mouse islets and MIN6 cells. We show that the imprinted 14q32 miRNA cluster contains 56 miRNAs, 32 of which are predicted to target the mRNAs of T1D autoantigens and 12 of which are glucose-sensitive. Using miRNA mimics and inhibitors, we confirmed that at least 7 of these miRNAs modulate the mRNA levels of the T1D autoantigens. Dicer1 knockdown significantly reduced the mRNA levels of all 3 autoantigens, further confirming the importance of miRNAs in this regulation. We conclude that miRNAs are involved in regulating the expression of the major T1D autoantigens.

  3. Elucidation of a C-rich signature motif in target mRNAs of RNA-binding protein TIAR.

    PubMed

    Kim, Henry S; Kuwano, Yuki; Zhan, Ming; Pullmann, Rudolf; Mazan-Mamczarz, Krystyna; Li, Huai; Kedersha, Nancy; Anderson, Paul; Wilce, Matthew C J; Gorospe, Myriam; Wilce, Jacqueline A

    2007-10-01

    The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3' untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, and used to elucidate a common signature motif present among TIAR target transcripts. The predicted TIAR motif was an unexpectedly cytosine-rich, 28- to 32-nucleotide-long element forming a stem and a loop of variable size with an additional side loop. The ability of TIAR to bind an RNA oligonucleotide with a representative C-rich TIAR motif sequence was verified in vitro using surface plasmon resonance. By this analysis, TIAR containing two or three RNA recognition domains (TIAR12 and TIAR123) showed low but significant binding to the C-rich sequence. In vivo, insertion of the C-rich motif into a heterologous reporter strongly suppressed its translation in cultured cells. Using this signature motif, an additional approximately 2,209 UniGene targets were identified (2.0% of the total UniGene database). A subset of specific mRNAs were validated by RNP IP analysis. Interestingly, in response to treatment with short-wavelength UV light (UVC), a stress agent causing DNA damage, each of these target mRNAs bearing C-rich motifs dissociated from TIAR. In turn, expression of the encoded proteins was elevated in a TIAR-dependent manner. In sum, we report the identification of a C-rich signature motif present in TIAR target mRNAs whose association with TIAR decreases following exposure to a stress-causing agent.

  4. Expression of CXCL4 and aquaporin 3 and 10 mRNAs in patients with otitis media with effusion.

    PubMed

    Jin, Zhe; Cha, Sung Ho; Choi, Yong-Sung; Kim, Young Il; Choi, Sun A; Yeo, Seung Geun

    2016-02-01

    Bacterial infections in children with underdeveloped Eustachian tubes are a major cause of otitis media with effusion (OEM), and persistent effusion in the middle ear in these patients is a major cause of surgical intervention. CXCL4 is associated with bacterial infection, and aquaporins 3 and 10 are associated with water metabolism. This study assessed the expression of mRNAs encoding CXCL-4 and aquaporins 3 and 10 in the effusion of pediatric OME patients, and the association of this expression with clinical manifestations. Levels of CXCL4 and aquaporin 3 and 10 mRNA were assayed by real-time RT-PCR in the middle ear effusion of 38 pediatric patients with OME requiring ventilation tube insertion. The relationships of these mRNA levels with the presence of bacteria; concomitant diseases such as allergic rhinitis, sinusitis, and adenoid disease; recurrence of OME; and number of ventilation tube insertions were evaluated. CXCL4 and aquaporin 3 and 10 mRNAs were expressed in middle ear effusion of all OME patients. CXCL-4 mRNA levels were significantly lower when bacteria were present and in patients with concomitant diseases (p<0.05 each). Levels of all three mRNAs were unrelated to OME recurrence or number of ventilation tube insertions (p>0.05 each). The levels of CXCL4 and aquaporin 10 mRNAs were significantly correlated (p<0.05). Expression of CXCL4 and aquaporin 3 and 10 mRNAs in middle ear effusion is associated with the pathophysiology of OME. CXCL4 mRNA levels are significantly lower in patients with than without concomitant diseases or bacterial infections. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Kinetics and intracellular compartmentalization of HTLV-1 gene expression: nuclear retention of HBZ mRNAs

    PubMed Central

    Rende, Francesca; Cavallari, Ilaria; Corradin, Alberto; Silic-Benussi, Micol; Toulza, Frederic; Toffolo, Gianna M.; Tanaka, Yuetsu; Jacobson, Steven; Taylor, Graham P.; D'Agostino, Donna M.; Bangham, Charles R. M.

    2011-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) codes for 9 alternatively spliced transcripts and 2 major regulatory proteins named Tax and Rex that function at the transcriptional and posttranscriptional levels, respectively. We investigated the temporal sequence of HTLV-1 gene expression in primary cells from infected patients using splice site-specific quantitative RT-PCR. The results indicated a two-phase kinetics with the tax/rex mRNA preceding expression of other viral transcripts. Analysis of mRNA compartmentalization in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of the two-phase kinetics and revealed strong nuclear retention of HBZ mRNAs, supporting their function as noncoding transcripts. Mathematical modeling underscored the importance of a delay between the functions of Tax and Rex, which was supported by experimental evidence of the longer half-life of Rex. These data provide evidence for a temporal pattern of HTLV-1 expression and reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts. PMID:21398577

  6. Effect of zinc deficiency of expression of specific mRNAs in rat liver

    SciTech Connect

    Chen, S.J.; Kimball, S.R.; Leure-duPree, A.E.; Jefferson, L.S. )

    1991-03-15

    Retinol is released from the liver bound to a specific transport protein, retinol binding protein (RBP), which binds to transthyretin (TTR) to transport retinol to the retinal pigment epithelium for use in the visual cycle. The synthesis of RBP as well as the transport of vitamin A from the liver is especially sensitive to zinc deficiency (ZD). Impaired hepatic synthesis of RBP has been reported in zinc-deficient rats. In the present study, the effect of ZD on the expression of mRNAs in the liver was examined by isolating total RNA from control, pair-fed, and zinc-deficient rats and translating the RNA in a messenger-dependent reticulocyte lysate. The radiolabeled translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The amounts of 12 of the approximately 200 radiolabeled translation products which could be distinguished were found to be altered in zinc-deficient compare to control samples. To investigate the expression of a specific mRNA, a cDNA to TTR was employed to probe the RNA samples. Slot blot analysis revealed that TTR mRNA was reduced to 57 {plus minus} 14% of the control in pair-fed rats to 29 {plus minus} 19% of control in zinc-deficient rats. The decrease in TTR mRNA is consistent with the observation that serum TTR is decreased during zinc deficiency caused by cirrhosis.

  7. Deadenylase depletion protects inherited mRNAs in primordial germ cells.

    PubMed

    Swartz, S Zachary; Reich, Adrian M; Oulhen, Nathalie; Raz, Tal; Milos, Patrice M; Campanale, Joseph P; Hamdoun, Amro; Wessel, Gary M

    2014-08-01

    A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

  8. Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

    PubMed

    Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

    1985-10-01

    Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

  9. An efficient system for selectively altering genetic information within mRNAs

    PubMed Central

    Montiel-González, Maria Fernanda; Vallecillo-Viejo, Isabel C.; Rosenthal, Joshua J. C.

    2016-01-01

    Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a UAG premature termination codon (PTC) to tryptophan (UGG) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool. PMID:27557710

  10. Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer.

    PubMed

    Rodríguez, Marta; Silva, Javier; Herrera, Alberto; Herrera, Mercedes; Peña, Cristina; Martín, Paloma; Gil-Calderón, Beatriz; Larriba, María Jesús; Coronado, M Josés; Soldevilla, Beatriz; Turrión, Víctor S; Provencio, Mariano; Sánchez, Antonio; Bonilla, Félix; García-Barberán, Vanesa

    2015-12-01

    Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a "stemness and metastatic" signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.

  11. Expression of metallothionein mRNAs on mouse cerebellum microglia cells by thimerosal and its metabolites.

    PubMed

    Minami, Takeshi; Miyata, Eriko; Sakamoto, Yamato; Kohama, Azusa; Yamazaki, Hideo; Ichida, Seiji

    2009-06-30

    Effects of thimerosal and its metabolites, ethyl mercury and thiosalicylate, on the expression of metallothionein (MT) mRNAs in mouse cerebellum microglia cell line, C8-B4 cells, were studied. The level of MT-1 mRNA significantly decreased at early hours and recovered time-dependently 24h after thimerosal was added to the C8-B4 cells. However, MT-2 and MT-3 mRNA expressions did not change from the control group. In contrast, the expression of MT-1 mRNA increased in a mouse neuroblastoma cell line 6h after incubation with thimerosal. In addition, the level of MT-1 mRNA decreased in C8-B4 cells 6h after the addition of thiosalicylate, but ethyl mercury induced MT-1 mRNA expression. When cell viability was compared with thimerosal, thiosalicylate, and ethyl mercury, the viability of C8-B4 cells decreased dose-dependently 24h after either thimerosal or ethyl mercury was added; however, the viability increased dose-dependently until 15 microM thiosalicylate was added. From the present results, it is concluded that the expression of MT-1 mRNA may be mediated by different factors than the expression of MT-2 mRNA in C8-B4 cells. The reduction of MT-1 mRNA level by thiosalicylate may affect the proliferation of C8-B4 cells.

  12. Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation.

    PubMed

    Bolduc, François; Garant, Jean-Michel; Allard, Félix; Perreault, Jean-Pierre

    2016-10-07

    G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5' and 3'-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed.

  13. Discovery of m7G-cap in eukaryotic mRNAs

    PubMed Central

    FURUICHI, Yasuhiro

    2015-01-01

    Terminal structure analysis of an insect cytoplasmic polyhedrosis virus (CPV) genome RNA in the early 1970s at the National Institute of Genetics in Japan yielded a 2′-O-methylated nucleotide in the 5′ end of double-stranded RNA genome. This finding prompted me to add S-adenosyl-L-methionine, a natural methylation donor, to the in vitro transcription reaction of viruses that contain RNA polymerase. This effort resulted in unprecedented mRNA synthesis that generates a unique blocked and methylated 5′ terminal structure (referred later to as “cap” or “m7G-cap”) in the transcription of silkworm CPV and human reovirus and vaccinia viruses that contain RNA polymerase in virus particles. Initial studies with viruses paved the way to discover the 5′-cap m7GpppNm structure present generally in cellular mRNAs of eukaryotes. I participated in those studies and was able to explain the pathway of cap synthesis and the significance of the 5′ cap (and capping) in gene expression processes, including transcription and protein synthesis. In this review article I concentrate on the description of these initial studies that eventually led us to a new paradigm of mRNA capping. PMID:26460318

  14. An efficient system for selectively altering genetic information within mRNAs.

    PubMed

    Montiel-González, Maria Fernanda; Vallecillo-Viejo, Isabel C; Rosenthal, Joshua J C

    2016-12-01

    Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a U A: G premature termination codon (PTC) to tryptophan (U G: G) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer

    PubMed Central

    Rodríguez, Marta; Silva, Javier; Herrera, Alberto; Herrera, Mercedes; Peña, Cristina; Martín, Paloma; Gil-Calderón, Beatriz; Larriba, María Jesús; Coronado, Mª José; Soldevilla, Beatriz; Turrión, Víctor S.; Provencio, Mariano; Sánchez, Antonio; Bonilla, Félix; García-Barberán, Vanesa

    2015-01-01

    Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a “stemness and metastatic” signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients. PMID:26528758

  16. Changes of adrenomedullin and its receptor components mRNAs expression in the brain stem and hypothalamus-pituitary-adrenal axis of stress-induced hypertensive rats.

    PubMed

    Li, Xia; Li, Liang; Shen, Lin-Lin; Qian, Yuan; Cao, Yin-Xiang; Zhu, Da-Nian

    2004-12-25

    In this study, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the changes in mRNAs levels of preproadrenomedullin (ppADM) gene encoding adrenomedullin (ADM) and the essential receptor components of ADM, calcitonin receptor-like receptor (CRLR), and the receptor activity modifying protein 2 and 3 (RAMP2 and RAMP3) in the medulla oblongata, hypothalamus, midbrain, pituitary gland and adrenal gland of the stress-induced hypertensive rats. It was shown that chronic foot-shock and noise stress for 15 consecutive days induced a significant increase in systolic blood pressure (SBP) and unique changes in ppADM and its receptor components mRNAs in all areas studied. As compared with the control group, the level of ppADM mRNA, normalized against a glyceraldehydes-3-phosphate dehydrogenase (GAPDH) control, was up-regulated in the hypothalamus-pituitary-adrenal (HPA) axis, but down-regulated in the medulla oblongata and midbrain (P<0.01 and P<0.05, respectively). The relative amount of CRLR mRNA was higher in the hypothalamus than that in other areas. The level of CRLR mRNA expression was significantly increased in the medulla oblongata of the stress group (P<0.01), but decreased in the midbrain (P<0.01) as well as hypothalamus(P<0.05), as compared with that of the control group. Chronic stress for 15 consecutive days produced an increase in the level of RAMP2 mRNA expression in the medulla oblongata (P<0.01) and a decrease in the adrenal gland (P<0.01), as compared with the control. No significant stress-related changes in RAMP2 mRNA were observed in the midbrain, hypothalamus and pituitary gland. The amount of RAMP3 mRNA was relatively higher in the midbrain and hypothalamus than that in the medulla oblongata, adrenal gland and adrenal gland. Stress-induced hypertensive rats exhibited an increased RAMP3 mRNA expression in the hypothalamus and pituitary gland (P<0.01 and P<0.05, respectively) and a decrease in the adrenal gland and midbrain (P<0

  17. Detection of early gene expression changes during activation of human primary lymphocytes by in vitro synthesis of proteins from polysome-associated mRNAs

    PubMed Central

    Miyamoto, Suzanne; Qin, Jun; Safer, Brian

    2001-01-01

    The rapid increase in protein synthesis during the mitogenic stimulation of human peripheral blood lymphocyte is the result of global and specific translational control mechanisms. To study some of these mechanisms, we examined the in vitro translatability of mRNAs associated with the polyribosome fraction. Polyribosome fractions were isolated from lymphocytes after activation with ionomycin and the phorbol ester PMA. The associated PAmRNAs were translated in the presence of mRNA-depleted rabbit reticulocyte lysate and [35S]Met, and the protein products were analyzed by SDS–PAGE and autoradiography. There was little synthesis of protein from the PAmRNAs isolated from unactivated T cells, but the PAmRNAs isolated from activated T cells showed a rapid increase in translatability. Translation of the PAmRNAs was sensitive to edeine and m7GTP, suggesting their cap-dependent translation. With activation, the majority of proteins showed increasing in vitro translation, but two proteins, p72 and p33, were found to have increased synthesis within 30 min, which decreased in 1 h. Transcription inhibitors were used to ascertain if regulation of their expression was transcriptional or translational. To identify these proteins, we used biotinylated lysine during the in vitro translation reaction, and we extracted the biotinylated protein by using streptavidin magnetic beads. The protein product was analyzed by mass spectrometry. p33 was identified as a prohibitin-like protein (BAP37), but the identification of p72 was not found in the databases. The distinct up-regulation and down-regulation of their protein expression suggest their tightly controlled regulation during early T cell activation. PMID:11266628

  18. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells

    PubMed Central

    MONTAGNER, GIULIA; GEMMO, CHIARA; FABBRI, ENRICA; MANICARDI, ALEX; ACCARDO, IGEA; BIANCHI, NICOLETTA; FINOTTI, ALESSIA; BREVEGLIERI, GIULIA; SALVATORI, FRANCESCA; BORGATTI, MONICA; LAMPRONTI, ILARIA; BRESCIANI, ALBERTO; ALTAMURA, SERGIO; CORRADINI, ROBERTO; GAMBARI, ROBERTO

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  19. Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation*

    PubMed Central

    Bolduc, François; Garant, Jean-Michel; Allard, Félix; Perreault, Jean-Pierre

    2016-01-01

    G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5′ and 3′-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo. Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed. PMID:27557661

  20. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

    PubMed Central

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-01-01

    AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

  1. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

    PubMed

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-02-15

    To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

  2. Norepinephrine triggers metaplasticity of LTP by increasing translation of specific mRNAs.

    PubMed

    Maity, Sabyasachi; Rah, Sean; Sonenberg, Nahum; Gkogkas, Christos G; Nguyen, Peter V

    2015-10-01

    Norepinephrine (NE) is a key modulator of synaptic plasticity in the hippocampus, a brain structure crucially involved in memory formation. NE boosts synaptic plasticity mostly through initiation of signaling cascades downstream from beta (β)-adrenergic receptors (β-ARs). Previous studies demonstrated that a β-adrenergic receptor agonist, isoproterenol, can modify the threshold for long-term potentiation (LTP), a putative cellular mechanism for learning and memory, in a process known as "metaplasticity." Metaplasticity is the ability of synaptic plasticity to be modified by prior experience. We asked whether NE itself could engage metaplastic mechanisms in area CA1 of mouse hippocampal slices. Using extracellular field potential recording and stimulation, we show that application of NE (10 µM), which did not alter basal synaptic strength, enhances the future maintenance of LTP elicited by subthreshold, high-frequency stimulation (HFS: 1 × 100 Hz, 1 sec). HFS applied 30 min after NE washout induced long-lasting (>4 h) LTP, which was significantly extended in duration relative to HFS alone. This NE-induced metaplasticity required β1-AR activation, as coapplication of the β1-receptor antagonist CGP-20712A (1 µM) attenuated maintenance of LTP. We also found that NE-mediated metaplasticity was translation- and transcription-dependent. Polysomal profiles of CA1 revealed increased translation rates for specific mRNAs during NE-induced metaplasticity. Thus, activation of β-ARs by NE primes synapses for future long-lasting plasticity on time scales extending beyond fast synaptic transmission; this may facilitate neural information processing and the subsequent formation of lasting memories. © 2015 Maity et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Double In situ Hybridization for MicroRNAs and mRNAs in Brain Tissues

    PubMed Central

    Kasai, Atsushi; Kakihara, Sora; Miura, Hiroki; Okada, Ryo; Hayata-Takano, Atsuko; Hazama, Keisuke; Niu, Misaki; Shintani, Norihito; Nakazawa, Takanobu; Hashimoto, Hitoshi

    2016-01-01

    MicroRNAs (miRNAs) participate in a variety of functions in the brain. Understanding the in vivo localization of miRNAs is an important step for uncovering their roles in brain function. However, the in situ detection of low-abundance miRNAs in brain tissues remains difficult and requires extensive optimization of in situ hybridization (ISH) protocols in individual laboratories. Thus, detailed information regarding experimental conditions would serve as a useful reference for researchers in this field. Here, we investigated and summarized the effects of adjusting a series of critical steps, including tissue fixation, probe accessibility and hybridization stringency, to standardize the currently used miRNA ISH procedures. As a result, we successfully detected several low-abundance miRNAs by ISH using the following experimental conditions: (1) use of fresh brain tissues, (2) digestion of brain samples with proteinase K, (3) LNA-probe hybridization at a temperature 37°C below the melting temperature of the RNA, (4) performance of high-stringency wash steps using 50% formamide in 1 × standard saline citrate (SSC) buffer. RT-PCR of the punched-out tissues using TaqManTM primers confirmed the ISH results. Finally, double-fluorescence ISH successfully demonstrated the colocalization of miRNAs and mRNAs. Thus, the detailed information regarding the miRNA ISH procedures used in this study may help to resolve the technical hurdles observed in the in vivo localization of miRNAs, and the elucidation of the specific roles of miRNAs. PMID:27920667

  4. Increased plasma mRNAs of placenta-specific 1 (PLAC1) and glial cells-missing 1 (GCM1) in mothers with pre-eclampsia.

    PubMed

    Fujito, Naoya; Samura, Osamu; Miharu, Norio; Tanigawa, Miho; Hyodo, Maki; Kudo, Yoshiki

    2006-03-01

    In this study we have investigated whether quantitative analysis of placental mRNAs in maternal plasma provides a way to monitor placental status. We measured plasma concentrations of human chorionic gonadotropin beta-subunit (betahCG) and human placental lactogen (hPL) mRNAs as previously reported mRNAs and pregnancy associated plasma protein A (PAPP-A), placenta-specific 1 (PLAC1) and glial cells-missing 1 (GCM1) mRNAs, which have not been measured during the course of normal pregnancy. Firstly, peripheral blood was obtained at various times from healthy pregnant women to clarify the time course of placental mRNAs. Secondly, blood was obtained from women with pre-eclampsia and gestational age-matched controls to examine whether placental mRNAs change in pre-eclampsia. Plasma was separated from these samples for extraction of RNA, followed by reverse transcription polymerse chain reaction analysis. Median concentrations of PLAC1 and GCM1 mRNA in plasma of pre-eclamptic subjects respectively were 1625 and 2141 copies/ml, significantly higher than 195 and 881 copies/ml, the values for controls (Mann-Whitney test, p<0.001). No significant difference was seen in hPL, betahCG, or PAPP-A mRNA concentration between pre-eclamptic and control groups. Plasma PLAC1 and GCM1 mRNAs appear promising as noninvasively measurable molecular markers for pre-eclampsia.

  5. Expressional potency of mRNAs encoding receptors and voltage-activated channels in the postmortem rat brain.

    PubMed Central

    Ragsdale, D S; Miledi, R

    1991-01-01

    The stability and integrity of mRNAs encoding neurotransmitter receptors and voltage-activated channels in the postmortem rat brain was investigated by isolating poly(A)+ mRNA, injecting it into Xenopus oocytes, and then examining the expression of functional neurotransmitter receptors and voltage-activated channels in the oocyte membrane by electrophysiological recording. This approach was also used to assess the stability of mRNAs in brains that were incubated in oxygenated mammalian Ringer's solution for various lengths of time and from brains that were freshly frozen and then thawed at room temperature. Oocytes injected with mRNA from up to 21-hr postmortem brains gave large agonist- and voltage-activated responses, indicating that mRNAs encoding neurotransmitter receptors and voltage-activated channels are relatively stable in postmortem brain tissue. In contrast, oocytes injected with mRNA from brains incubated in Ringer's solution exhibited smaller responses, and oocytes injected with mRNA from tissue that was frozen and then thawed displayed very small or undetectable responses. Northern blot analysis using a nucleic acid probe for rat brain Na(+)-channel mRNA indicated that the size of the Na+ currents in injected oocytes reflected the levels of mRNA for Na+ channels in the different mRNA preparations. Thus, the expressional potency of mRNAs encoding neurotransmitter receptors and voltage-activated channels is quite stable in postmortem brains in situ, but it is reduced if the brains are kept in oxygenated saline, and freezing and thawing of tissue results in rapid degeneration of mRNA. Images PMID:1705710

  6. Rapid Degradation of Host mRNAs by Stimulation of RNase E Activity by Srd of Bacteriophage T4

    PubMed Central

    Qi, Dan; Alawneh, Abdulraheem M.; Yonesaki, Tetsuro; Otsuka, Yuichi

    2015-01-01

    Escherichia coli messenger RNAs (mRNAs) are rapidly degraded immediately after bacteriophage T4 infection, and the host RNase E contributes to this process. Here, we found that a previously uncharacterized factor of T4 phage, Srd (Similarity with rpoD), was involved in T4-induced host mRNA degradation. The rapid decay of ompA and lpp mRNAs was partially alleviated and a decay intermediate of lpp mRNA rapidly accumulated in cells infected with T4 phage lacking srd. Exogenous expression of Srd in uninfected cells significantly accelerated the decay of these mRNAs. In addition, lpp(T) RNA, with a sequence identical to the decay intermediate of lpp mRNA and a triphosphate at 5′-end, was also destabilized by Srd. The destabilization of these RNAs by Srd was not observed in RNase E-defective cells. The initial cleavage of a primary transcript by RNase E can be either direct or dependent on the 5′-end of transcript. In the latter case, host RppH is required to convert the triphosphate at 5′-end to a monophosphate. lpp(T) RNA, but not lpp and ompA mRNAs, required RppH for Srd-stimulated degradation, indicating that Srd stimulates both 5′-end-dependent and -independent cleavage activities of RNase E. Furthermore, pull-down and immunoprecipitation analyses strongly suggested that Srd physically associates with the N-terminal half of RNase E containing the catalytic moiety and the membrane target sequence. Finally, the growth of T4 phage was significantly decreased by the disruption of srd. These results strongly suggest that the stimulation of RNase E activity by T4 Srd is required for efficient phage growth. PMID:26323881

  7. Rhn1, a nuclear protein, is required for suppression of meiotic mRNAs in mitotically dividing fission yeast.

    PubMed

    Sugiyama, Tomoyasu; Sugioka-Sugiyama, Rie; Hada, Kazumasa; Niwa, Ryusuke

    2012-01-01

    In the fission yeast Schizosaccharomyces pombe, many meiotic mRNAs are transcribed during mitosis and meiosis and selectively eliminated in mitotic cells. However, this pathway for mRNA decay, called the determinant of selective removal (DSR)-Mmi1 system, targets only some of the numerous meiotic mRNAs that are transcribed in mitotic cells. Here we describe Rhn1, a nuclear protein involved in meiotic mRNA suppression in vegetative fission yeast. Rhn1 is homologous to budding yeast Rtt103 and localizes to one or a few discrete nuclear dots in growing vegetative cells. Rhn1 colocalizes with a pre-mRNA 3'-end processing factor, Pcf11, and with the 5'-3' exoribonuclease, Dhp1; moreover, Rhn1 coimmunoprecipitates with Pcf11. Loss of rhn1 results in elevated sensitivity to high temperature, to thiabendazole (TBZ), and to UV. Interestingly, meiotic mRNAs--including moa1(+), mcp5(+), and mug96(+)--accumulate in mitotic rhn1Δ cells. Accumulation of meiotic mRNAs also occurs in strains lacking Lsk1, a kinase that phosphorylates serine 2 (Ser-2) in the C-terminal domain (CTD) of RNA polymerase II (Pol II), and in strains lacking Sen1, an ATP-dependent 5'-3' RNA/DNA helicase: notably, both Lsk1 and Sen1 have been implicated in termination of Pol II-dependent transcription. Furthermore, RNAi knockdown of cids-2, a Caenorhabditis elegans ortholog of rhn1(+), leads to elevated expression of a germline-specific gene, pgl-1, in somatic cells. These results indicate that Rhn1 contributes to the suppression of meiotic mRNAs in vegetative fission yeast and that the mechanism by which Rhn1 downregulates germline-specific transcripts may be conserved in unicellular and multicellular organisms.

  8. Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific.

    PubMed

    Wang, Tong; Cui, Yizhi; Jin, Jingjie; Guo, Jiahui; Wang, Guibin; Yin, Xingfeng; He, Qing-Yu; Zhang, Gong

    2013-05-01

    As a well-known phenomenon, total mRNAs poorly correlate to proteins in their abundances as reported. Recent findings calculated with bivariate models suggested even poorer such correlation, whereas focusing on the translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) subset. In this study, we analysed the relative abundances of mRNAs, RNC-mRNAs and proteins on genome-wide scale, comparing human lung cancer A549 and H1299 cells with normal human bronchial epithelial (HBE) cells, respectively. As discovered, a strong correlation between RNC-mRNAs and proteins in their relative abundances could be established through a multivariate linear model by integrating the mRNA length as a key factor. The R(2) reached 0.94 and 0.97 in A549 versus HBE and H1299 versus HBE comparisons, respectively. This correlation highlighted that the mRNA length significantly contributes to the translational modulation, especially to the translational initiation, favoured by its correlation with the mRNA translation ratio (TR) as observed. We found TR is highly phenotype specific, which was substantiated by both pathway analysis and biased TRs of the splice variants of BDP1 gene, which is a key transcription factor of transfer RNAs. These findings revealed, for the first time, the intrinsic and genome-wide translation modulations at translatomic level in human cells at steady-state, which are tightly correlated to the protein abundance and functionally relevant to cellular phenotypes.

  9. TH and DCX mRNAs in peripheral blood and bone marrow predict outcome in metastatic neuroblastoma patients.

    PubMed

    Yáñez, Yania; Hervás, David; Grau, Elena; Oltra, Silvestre; Pérez, Gema; Palanca, Sarai; Bermúdez, Mar; Márquez, Catalina; Cañete, Adela; Castel, Victoria

    2016-03-01

    In metastatic neuroblastoma (NB) patients, accurate risk stratification and disease monitoring would reduce relapse probabilities. This study aims to evaluate the independent prognostic significance of detecting tyrosine hydroxylase (TH) and doublecortin (DCX) mRNAs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral blood (PB) and bone marrow (BM) samples from metastatic NB patients. RT-qPCR was performed on PB and BM samples from metastatic NB patients at diagnosis, post-induction therapy and at the end of treatment for TH and DCX mRNAs detection. High levels of TH and DCX mRNAs when detected in PB and BM at diagnosis independently predicted worse outcome in a cohort of 162 metastatic NB. In the subgroup of high-risk metastatic NB, TH mRNA detected in PB remained as independent predictor of EFS and OS at diagnosis. After the induction therapy, high levels of TH mRNA in PB and DCX mRNA in BM independently predicted poor EFS and OS. Furthermore TH mRNA when detected in BM predicted worse EFS. TH mRNA in PB samples at the end of treatment is an independent predictor of worse outcome. TH and DCX mRNAs levels in PB and BM assessed by RT-qPCR should be considered in new pre-treatment risk stratification strategies to reliable estimate outcome differences in metastatic NB patients. In those high-risk metastatic NB, TH and DCX mRNA quantification could be used for the assessment of response to treatment and for early detection of progressive disease or relapses.

  10. RNA sequencing and proteogenomics reveal the importance of leaderless mRNAs in the radiation-tolerant bacterium Deinococcus deserti.

    PubMed

    de Groot, Arjan; Roche, David; Fernandez, Bernard; Ludanyi, Monika; Cruveiller, Stéphane; Pignol, David; Vallenet, David; Armengaud, Jean; Blanchard, Laurence

    2014-04-01

    Deinococcus deserti is a desiccation- and radiation-tolerant desert bacterium. Differential RNA sequencing (RNA-seq) was performed to explore the specificities of its transcriptome. Strikingly, for 1,174 (60%) mRNAs, the transcription start site was found exactly at (916 cases, 47%) or very close to the translation initiation codon AUG or GUG. Such proportion of leaderless mRNAs, which may resemble ancestral mRNAs, is unprecedented for a bacterial species. Proteomics showed that leaderless mRNAs are efficiently translated in D. deserti. Interestingly, we also found 173 additional transcripts with a 5'-AUG or 5'-GUG that would make them competent for ribosome binding and translation into novel small polypeptides. Fourteen of these are predicted to be leader peptides involved in transcription attenuation. Another 30 correlated with new gene predictions and/or showed conservation with annotated and nonannotated genes in other Deinococcus species, and five of these novel polypeptides were indeed detected by mass spectrometry. The data also allowed reannotation of the start codon position of 257 genes, including several DNA repair genes. Moreover, several novel highly radiation-induced genes were found, and their potential roles are discussed. On the basis of our RNA-seq and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single-nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions.

  11. Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus

    SciTech Connect

    Inglis, S.C.

    1982-12-01

    Cloned DNA copies of two cellular genes were used to monitor, by blot hybridization, the stability of particular cell mRNAs after infection by influenza virus and herpes virus. The results indicated that the inhibition of host cell protein synthesis that accompanied infection by each virus could be explained by a reduction in the amounts of cellular mRN As in the cytoplasm, and they suggested that this decrease was due to virus-mediated mRNA degradation.

  12. Stromal expression of 72 kda type IV collagenase (MMP-2) and TIMP-2 mRNAs in colorectal neoplasia.

    PubMed Central

    Poulsom, R.; Pignatelli, M.; Stetler-Stevenson, W. G.; Liotta, L. A.; Wright, P. A.; Jeffery, R. E.; Longcroft, J. M.; Rogers, L.; Stamp, G. W.

    1992-01-01

    We undertook an in situ hybridization study to localize the mRNAs for the 72 kda type IV collagenase (MMP-2) and its specific inhibitor (TIMP-2) in 12 colorectal carcinomas, 3 adenomas, and 4 uninvolved resection margins to see how their distributions correlated with that of the reported distribution of MMP-2 protein. Labeling for MMP-2 and TIMP-2 mRNAs was detectable in 10 of 12 carcinomas and in 2 of 3 adenomas. Unexpectedly, we found much stronger signals for MMP-2 and TIMP-2 mRNAs within the mesenchymal cells in the desmoplastic stroma, of endothelial and/or (myo)fibroblastic nature, rather than in tumor epithelial cells in which localization of MMP-2 was anticipated. Our data indicate that stromal cells may have the ability to synthesize a metalloproteinase that degrades basement membrane, and may together with the neoplastic epithelial cells participate actively in the tissue remodeling and disruption of the basement membrane integrity which is characteristic of invasive tumors. Images Figure 1 to 6 PMID:1323219

  13. Isolation of a trans-acting factor involved in localization of Paracentrotus lividus maternal mRNAs.

    PubMed Central

    Costa, C; Romancino, D P; Ingrassia, A; Vizzini, A; Di Carlo, M

    1999-01-01

    Localization of Paracentrotus lividus bep maternal mRNAs at the animal pole occurs by association with the cytoskeleton and involves a 54-kDa protein, called LP54, that is able to bind to the 3' untranslated regions (UTRs) of bep mRNAs. We describe here the isolation and purification of this protein. Antibodies raised against purified LP54 allowed us to establish its localization in P. lividus eggs and embryos. This localization coincides with the mRNAs with which it is associated, that is, the animal pole in the egg, and, after fertilization, the regions derived from this part of the egg, and finally the oral ectoderm of the pluteus. Association with the cytoskeleton was shown by the copurification of LP54 in a microtubule preparation. Involvement in bep mRNA localization was demonstrated by microinjection of anti-LP54 antibodies in P. lividus eggs, which caused alteration of spatial distribution of bep3 mRNA. PMID:10573120

  14. Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes.

    PubMed

    Li, Chunliang; Qi, Rong; Singleterry, Rebecca; Hyle, Judith; Balch, Amanda; Li, Xiuling; Sublett, Jack; Berns, Hartmut; Valentine, Marcus; Valentine, Virginia; Sherr, Charles J

    2014-05-01

    Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB × C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to "spacer" sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic coinjection of TALEN mRNAs directed to the Agouti, miR-205, and the Arf tumor suppressor loci yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knockout technology in embryonic stem cells.

  15. Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli.

    PubMed

    Benhalevy, Daniel; Bochkareva, Elena S; Biran, Ido; Bibi, Eitan

    2015-01-01

    Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

  16. Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation.

    PubMed

    Schneider, Nils; Meier, Matthias

    2017-02-01

    Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA-DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications. © 2017 Schneider and Meier; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation

    PubMed Central

    Zhu, Yiping; Chen, Guifang; Lv, Fengxiang; Wang, Xinlu; Ji, Xin; Xu, Yihui; Sun, Jing; Wu, Li; Zheng, Yong-Tang; Gao, Guangxia

    2011-01-01

    The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 3′ end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5′ end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5′ and 3′ mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs. PMID:21876179

  18. Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM)

    PubMed Central

    1986-01-01

    Rabbit polyclonal antibodies directed against the chicken neural cell adhesion molecule (N-CAM) were used to isolate four overlapping cDNA clones from a chicken cDNA expression library in bacteriophage gamma gt11. These clones collectively accounted for 3.8 kilobases of N-CAM mRNA sequence and hybridized specifically to two 6-7-kilobase brain polyadenylated RNA species that co-migrated with previously identified N-CAM mRNAs. DNA fragments derived from an internal region of the cloned cDNA sequences hybridized to the larger but not to the smaller N- CAM mRNA species, while fragments on either side of this region hybridized to both mRNAs. A cDNA fragment that recognized only the larger mRNA was subcloned into gamma gt11, and the expressed fusion protein was used to affinity-purify rabbit polyclonal antibodies; the antibodies recognized only the larger of the two structurally related N- CAM polypeptides. In contrast, when several cDNA clones that recognized both mRNAs were used to purify antibodies, the antibodies recognized both polypeptides. The results, in conjunction with other data indicating that there is one gene specifying N-CAM, suggest that different N-CAM polypeptides are synthesized from multiple N-CAM messages generated by alternative splicing of transcripts from a single N-CAM gene. PMID:3001098

  19. High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs

    PubMed Central

    Endo, Kei; Hayashi, Karin; Saito, Hirohide

    2016-01-01

    The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations. PMID:26902536

  20. Identification of mRNAs differentially expressed in quiescence or in late G1 phase of the cell cycle in human breast cancer cells by using the differential display method.

    PubMed Central

    Alpan, R. S.; Sparvero, S.; Pardee, A. B.

    1996-01-01

    BACKGROUND: The decision for a cell to enter the DNA synthesis (S) phase of the cell cycle or to arrest in quiescence is likely to be determined by genes expressed in the late G1 phase, at the restriction point. Loss of restriction point control is associated with malignant cellular transformation and cancer. For this reason, identifying genes that are differentially expressed in late G1 phase versus quiescence is important for understanding the molecular basis of normal and malignant growth. MATERIALS AND METHODS: The differential display (DD) method detects mRNA species that are different between sets of mammalian cells, allowing their recovery and cloning of the corresponding cDNAs. Using this technique, we compared mRNAs from synchronized human breast cancer cells (21 PT) in quiescence and in late G1. RESULTS: Six mRNAs differentially expressed in late G1 or in quiescence were identified. One mRNA expressed 10 hr after serum induction showed 99% homology to a peptide transporter involved in antigen presentation of the class I major histocompatibility complex (TAP-1) mRNA. Another mRNA expressed specifically in quiescence and down-regulated 2 hr following serum induction showed 98% homology to human NADP+ -dependent cytoplasmic malic enzyme (EC1.1.1.40) mRNA, which is an important enzyme in fatty acid synthesis and lipogenesis. Three others showed high homology to different mRNAs in the GeneBank, corresponding to genes having unknown functions. Finally, one mRNA revealed no significant homology to known genes in the GeneBank. CONCLUSIONS: We conclude that DD is an efficient and powerful method for the identification of growth-related genes which may have a role in cancer development. Images FIG. 2 FIG. 3 PMID:8827717

  1. Selenium status highly-regulates selenoprotein mRNA levels for only a subset of the selenoproteins in the selenoproteome

    PubMed Central

    SUNDE, Roger A.; RAINES, Anna M.; BARNES, Kimberly M.; EVENSON, Jacqueline K.

    2011-01-01

    Synopsis Gpx (glutathione peroxidase)-1 enzyme activity and mRNA levels decrease dramatically in selenium (Se) deficiency, whereas other selenoproteins are less affected by Se deficiency. This hierarchy of Se regulation is not understood, but the position of the UGA selenocysteine codon is thought to play a major role in making selenoprotein mRNAs susceptible to nonsense-mediated decay. Thus in the present paper we studied the complete selenoproteome in the mouse to uncover additional selenoprotein mRNAs that are highly-regulated by Se status. Mice were fed Se-deficient, Se-marginal, and Se-adequate diets (0, 0.05 and 0.2 μg Se/g, respectively) for 35 days, and selenoprotein mRNA levels in liver and kidney were determined using microarray analysis and quantitative real-time PCR analysis. Se-deficient mice had liver Se concentrations and liver Gpx1 and thioredoxin reductase activities that were 4, 3 and 3%, respectively, of the levels in Se-adequate mice, indicating that the mice were Se-deficient. mRNAs for Selh (selenoprotein H) and Sepw1 (selenoprotein W) as well as Gpx1 were decreased by Se deficiency to <40% of Se-adequate levels. Five and two additional mRNAs were moderately down-regulated in Se-deficient liver and kidney, respectively. Importantly, nine selenoprotein mRNAs in liver and fifteen selenoprotein mRNAs in kidney were not significantly regulated by Se deficiency, clearly demonstrating that Se regulation of selenoprotein mRNAs is not a general phenomenon. The similarity of the response to Se deficiency suggests that there is one underlying mechanism responsible. Importantly, the position of the UGA codon did not predict susceptibility to Se regulation, clearly indicating that additional features are involved in causing selenoprotein mRNAs to be sensitive to Se status. PMID:19076066

  2. Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

    PubMed Central

    Cao, Yi; Li, Jie; Jiang, Na

    2014-01-01

    Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5′ UTR was present in the pta-ackA transcript. Removal of the 5′ UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5′ UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15. PMID:24317083

  3. Overlapping cis sites used for splicing of HIV-1 env/nef and rev mRNAs.

    PubMed

    Swanson, A K; Stoltzfus, C M

    1998-12-18

    Alternative splicing is used to generate more than 30 human immunodeficiency virus type 1 (HIV-1) spliced and unspliced mRNAs from a single primary transcript. The abundance of HIV-1 mRNAs is determined by the efficiencies with which its different 5' and 3' splice sites are used. Three splice sites (A4c, A4a, and A4b) are upstream of the rev initiator AUG. RNAs spliced at A4c, A4a, and A4b are used as mRNAs for Rev. Another 3' splice site (A5) is immediately downstream of the rev initiator. RNAs spliced at A5 are used as mRNAs for Env and Nef. In this report, primer extension analysis of splicing intermediates was used to show that there are eight branch points in this region, all of which map to adenosine residues. In addition, cis elements recognized by the cellular splicing machinery overlap; the two most 3' branch points overlap with the AG dinucleotides at rev 3' splice sites A4a and A4b. Competition of the overlapping cis sites for different splicing factors may play a role in maintaining the appropriate balance of mRNAs in HIV-1-infected cells. In support of this possibility, mutations at rev 3' splice site A4b AG dinucleotide dramatically increased splicing of the env/nef 3' splice site A5. This correlated with increased usage of the four most 3' branch points, which include those within the rev 3' splice site AG dinucleotides. Consistent with these results, analysis of a mutant in which three of the four env/nef branch points were inactivated indicated that use of splice site A5 was inhibited and splicing was shifted predominantly to the most 5' rev 3' splice site A4c with preferential use of the two most 5' branch points. Our results suggest that spliceosomes formed at rev A4a-4b, rev A4c, and env/nef A5 3' splice sites each recognize different subsets of the eight branch point sequences.

  4. Mechanism for stabilizing mRNAs involved in methanol-dependent methanogenesis of cold-adaptive Methanosarcina mazei zm-15.

    PubMed

    Cao, Yi; Li, Jie; Jiang, Na; Dong, Xiuzhu

    2014-02-01

    Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5' untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5' UTR was present in the pta-ackA transcript. Removal of the 5' UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5' UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5' UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15.

  5. Identification and characterization of antioxidant enzyme mRNAs in the rat epididymis.

    PubMed

    Zini, A; Schlegel, P N

    1997-04-01

    Spermatozoa are highly sensitive to oxidative stress. The epididymis, a natural sperm reservoir, has maturational and storage functions. The epididymis may also protect spermatozoa from oxidative injury by elaborating scavengers of reactive oxygen species (ROS). Therefore, we have evaluated the mRNA expression of antioxidant enzymes in the normal rat epididymis and the effects of efferent duct ligation no the expression of these enzymes. Adult rat epididymides were harvested, divided into caput, corpus and cauda and processed for RNA extraction. Additional adult rats were subjected to unilateral efferent duct ligation and the epididymides harvested at 1, 4, 8, 16 or 28 days after the procedure. Antioxidant enzyme mRNA expression was assessed by Northern blot analysis using 32P-labelled DNA probes derived from known cDNA sequences for classical cellular glutathione peroxidase (GSHPx), phospholipid hydroperoxide glutathione peroxidase (PHGPX), secretory epididymal glutathione peroxidase (E-GPX), copper-zinc superoxide dismutase (SOD), secretory epididymal superoxide dismutase (E-SOD) and catalase. Specific mRNA levels were measured, with gene expression evaluated relative to total RNA, not per organ. Variations in lane loading were controlled by measuring the levels of 28S ribosomal RNA. GSHPx, PHGPX, SOD and catalase mRNA were detected in the caput, corpus and cauda epididymis. E-GPX mRNA was only detected in the caput, whereas E-SOD mRNA was primarily detected in the corpus. At 28 days after efferent duct ligation, epididymal weight decreased by 34% relative to controls (p < 0.05). With the exception of PHGPX, the relative mRNA levels of the antioxidant enzymes studied did not change after efferent duct ligation. This study demonstrates that mRNAs for multiple antioxidant enzymes are expressed in the epididymis and that the relative expression of these enzymes remains largely unchanged in response to efferent duct ligation. Taken together, these results suggest

  6. Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs

    PubMed Central

    Pachernegg, Svenja; Joshi, Illah; Muth-Köhne, Elke; Pahl, Steffen; Münster, Yvonne; Terhag, Jan; Karus, Michael; Werner, Markus; Ma-Högemeier, Zhan-Lu; Körber, Christoph; Grunwald, Thomas; Faissner, Andreas; Wiese, Stefan; Hollmann, Michael

    2013-01-01

    Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs. PMID:24348335

  7. Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs

    PubMed Central

    Mumbach, Maxwell R.; Wurtzel, Omri; Sorek, Rotem

    2013-01-01

    The presence of 5-methylcytidine (m5C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis) and gram negative (E. coli) bacteria, an archaeon (S. solfataricus) and a eukaryote (S. cerevisiae), followed by massively parallel sequencing. We were able to recover most previously documented m5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m5C was absent were also discovered. Intriguingly, we detected m5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain. PMID:23825970

  8. mRNAs and miRNAs profiling of mesenchymal stem cells derived from amniotic fluid and skin: the double face of the coin.

    PubMed

    Lazzarini, Raffaella; Olivieri, Fabiola; Ferretti, Concetta; Mattioli-Belmonte, Monica; Di Primio, Roberto; Orciani, Monia

    2014-01-01

    Mesenchymal stem cells (MSCs) can be isolated from different adult sources and, even if the minimal criteria for defining MSCs have been reported, the scientific question about the potential distinctions among MSCs derived from different sources is still open. In particular, it is debated whether MSCs of different origin have the same grade of stemness or whether the source affects their undifferentiated status. Here, we report not only the isolation and the traditional characterization of MSCs derived from amniotic fluid (AF-MSCs) and skin (S-MSCs) but also a molecular characterization based on mRNAs and miRNAs profiling. Our results show that, even if both AF- and S-MSCs are mostly regulated by the same pathways (such as Wnt, MAPK and TGF-β), there are some important differences at the molecular level that directly affect important cellular features, such as the ability to differentiate into adipocytes. In conclusion, even if further studies are necessary to improve the knowledge about the role of each dysregulated miRNAs gene, these differences may actually strengthen the question about the importance of tissue origin.

  9. Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

    PubMed Central

    Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

    2010-01-01

    Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

  10. Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse.

    PubMed

    Filippini, Alice; Bonini, Daniela; Lacoux, Caroline; Pacini, Laura; Zingariello, Maria; Sancillo, Laura; Bosisio, Daniela; Salvi, Valentina; Mingardi, Jessica; La Via, Luca; Zalfa, Francesca; Bagni, Claudia; Barbon, Alessandro

    2017-06-22

    The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes.

  11. Cloning of growth hormone, somatolactin, and their receptor mRNAs, their expression in organs, during development, and on salinity stress in the hermaphroditic fish, Kryptolebias marmoratus.

    PubMed

    Rhee, Jae-Sung; Kim, Bo-Mi; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

    2012-04-01

    Salinity is an important parameter that affects survival and metabolism in fish. In fish, pituitary growth hormone (GH) regulates physiological functions including adaptation to different salinity as well as somatic growth. GH is stimulated by growth hormone-releasing hormone (GHRH) and exerts its function via binding to growth hormone receptor (GHR). As Kryptolebias marmoratus is a euryhaline fish, this species would be a useful model species for studying the adaptation to osmotic stress conditions. Here, we cloned GH, -GHR, somatolactin (SL), and somatolactin receptor (SLR) genes, and analyzed their expression patterns in different tissues and during early developmental stages by using real-time RT-PCR. We also further examined expression of them after acclimation to different salinity. Tissue distribution studies revealed that Km-GH and -SL mRNAs were remarkably expressed in brain and pituitary, whereas Km-GHR and -SLR mRNAs were predominantly expressed in liver, followed by gonad, muscle, pituitary, and brain. During embryonic developmental stages, the expression of their mRNA was increased at stage 3 (9 dpf). The Km-GH and -SL mRNA transcripts were constantly elevated until stage 5 (5h post hatch), whereas Km-GHR and -SLR mRNA levels decreased at this stage. After we transferred K. marmoratus from control (12 psu) to hyper-osmotic condition (hyperseawater, HSW; 33 psu), Km-GH, -SL, and GHR mRNA levels were enhanced. In hypo-osmotic conditions like freshwater (FW), Km-GH and -SL expressions were modulated 24 h after exposure, and Km-SLR transcripts were significantly upregulated. This finding suggests that Km-GH and -SL may be involved in the osmoregulatory mechanism under hyper-osmotic as well as hypo-osmotic stress. This is the first report on transcriptional modulation and relationship of GH, GHR, SL, and SLR during early development and after salinity stress. This study will be helpful to a better understanding on molecular mechanisms of adaptation response

  12. Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

    PubMed

    Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

    1993-01-01

    Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

  13. Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA.

    PubMed

    Gioio, Anthony E; Lavina, Zeno Scotto; Jurkovicova, Dana; Zhang, Hengshan; Eyman, Maria; Giuditta, Antonio; Kaplan, Barry B

    2004-08-01

    It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen. The results of this screen facilitated the identification of 31 mRNAs that encoded cytoskeletal proteins, translation factors, ribosomal proteins, molecular motors, metabolic enzymes, nuclear-encoded mitochondrial mRNAs, and a molecular chaperone. Results of cell fractionation and RT-PCR analyses established that several of these mRNAs were present in polysomes present in the presynaptic nerve terminal of photoreceptor neurons, indicating that these mRNAs were being actively translated. Findings derived from in vitro transfection studies established that these isolated nerve terminals had the ability to translate a heterologous reporter mRNA. Based upon these data, it is hypothesized that the local protein synthetic system plays an important role in the maintenance/remodelling of the cytoarchitecture of the axon and nerve terminal, maintenance of the axon transport and mRNA translation systems, as well as contributing to the viability and function of the local mitochondria.

  14. Translational regulation in nutrigenomics.

    PubMed

    Liu, Botao; Qian, Shu-Bing

    2011-11-01

    The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health.

  15. Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

    PubMed Central

    1987-01-01

    Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross- hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth. PMID:3558480

  16. Structural basis for ArfA-RF2-mediated translation termination on mRNAs lacking stop codons.

    PubMed

    Huter, Paul; Müller, Claudia; Beckert, Bertrand; Arenz, Stefan; Berninghausen, Otto; Beckmann, Roland; Wilson, Daniel N

    2017-01-26

    In bacteria, ribosomes stalled on truncated mRNAs that lack a stop codon are rescued by the transfer-messenger RNA (tmRNA), alternative rescue factor A (ArfA) or ArfB systems. Although tmRNA-ribosome and ArfB-ribosome structures have been determined, how ArfA recognizes the presence of truncated mRNAs and recruits the canonical termination release factor RF2 to rescue the stalled ribosomes is unclear. Here we present a cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2. The structure shows that the C terminus of ArfA binds within the mRNA entry channel on the small ribosomal subunit, and explains how ArfA distinguishes between ribosomes that bear truncated or full-length mRNAs. The N terminus of ArfA establishes several interactions with the decoding domain of RF2, and this finding illustrates how ArfA recruits RF2 to the stalled ribosome. Furthermore, ArfA is shown to stabilize a unique conformation of the switch loop of RF2, which mimics the canonical translation termination state by directing the catalytically important GGQ motif within domain 3 of RF2 towards the peptidyl-transferase centre of the ribosome. Thus, our structure reveals not only how ArfA recruits RF2 to the ribosome but also how it promotes an active conformation of RF2 to enable translation termination in the absence of a stop codon.

  17. Modification of tRNA(Lys) UUU by elongator is essential for efficient translation of stress mRNAs.

    PubMed

    Fernández-Vázquez, Jorge; Vargas-Pérez, Itzel; Sansó, Miriam; Buhne, Karin; Carmona, Mercè; Paulo, Esther; Hermand, Damien; Rodríguez-Gabriel, Miguel; Ayté, José; Leidel, Sebastian; Hidalgo, Elena

    2013-01-01

    The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNA(Lys) UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery.

  18. Modification of tRNALys UUU by Elongator Is Essential for Efficient Translation of Stress mRNAs

    PubMed Central

    Sansó, Miriam; Buhne, Karin; Carmona, Mercè; Paulo, Esther; Hermand, Damien; Rodríguez-Gabriel, Miguel; Ayté, José; Leidel, Sebastian; Hidalgo, Elena

    2013-01-01

    The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNALys UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery. PMID:23874237

  19. Activity-dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain.

    PubMed

    Tiruchinapalli, Dhanrajan M; Caron, Marc G; Keene, Jack D

    2008-12-01

    Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA-binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p-glycogen synthase kinase 3beta (GSK3beta) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIalpha, vascular early response gene, GAP-43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3beta signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3beta, p-Akt, and beta-catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile-X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.

  20. Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

    PubMed Central

    Kumar, Parimal; Hellen, Christopher U.T.; Pestova, Tatyana V.

    2016-01-01

    Ribosomal attachment to mammalian capped mRNAs is achieved through the cap–eukaryotic initiation factor 4E (eIF4E)–eIF4G–eIF3–40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5′-terminal AUGs was stimulated by the eIF4E–cap interaction and followed “the first AUG” rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5′ end of mRNA, implying that Met-tRNAiMet inspects mRNA from the first nucleotide and that initiation does not have a “blind spot.” In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E–eIF4G–eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E–eIF4G–eIF3–40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAiMet can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m7G cap specifically interacts with eIF3l. PMID:27401559

  1. Characterization and differential expression patterns of conserved microRNAs and mRNAs in three genders of the rice field eel (Monopterus albus).

    PubMed

    Gao, Yu; Guo, Wei; Hu, Qing; Zou, Ming; Tang, Rong; Chi, Wei; Li, Dapeng

    2014-01-01

    MicroRNAs (miRNAs) are endogenous small RNAs that can regulate target mRNAs by binding to their sequences in the 3' untranslated region. The expression of miRNAs and their biogenetic pathway are involved in sexual differentiation and in the regulation of the development of germ cells and gonadal somatic cells. The rice field eel (Monopterus albus) undergoes a natural sexual transformation from female to male via an intersex stage during its life cycle. To investigate the molecular mechanisms of this sexual transformation, miRNAs present in the different sexual stages of the rice field eel were identified by high-throughput sequencing technology. A significantly differential expression among the 3 genders (p < 0.001) was observed for 48 unique miRNAs and 3 miRNAs*. Only 9 unique miRNAs showed a more than 8-fold change in their expression among the 3 genders, including mal-miR-430a and mal-miR-430c which were higher in females than in males. However, mal-miR-430b was only detected in males. Several potential miRNA target genes (cyp19a, cyp19b, nr5a1b, foxl2 amh, and vasa) were also investigated. Real-time RT-PCR demonstrated highly specific expression patterns of these genes in the 3 genders of the rice field eel. Many of these genes are targets of mal-miR-430b according to the TargetScan and miRTarBase. These results suggest that the miR-430 family may be involved in the sexual transformation of the rice field eel.

  2. Quantitative aspects of gene regulation by small RNAs

    NASA Astrophysics Data System (ADS)

    Mehta, Pankaj

    2007-03-01

    Small, non-coding RNAs (sRNAs) play an important role as genetic regulators in both prokaryotes and eukaryotes. Many sRNAs act through base-pairing interaction with target messenger RNAs (mRNAs) to regulate transcription, translation, and mRNA stability. sRNAs represent a novel form of genetic regulation distinct from more thoroughly studied protein regulators. This talk addresses quantitative aspectsof sRNA-mediated genetic regulation, focusing on noise, tunability, and feedback. In particular, we compare and contrast sRNA and protein regulators in an attempt to understand the compartive advantages of each form of regulation.

  3. Selective translational control and nonspecific posttranscriptional regulation of ribosomal protein gene expression during development and regeneration of rat liver.

    PubMed Central

    Aloni, R; Peleg, D; Meyuhas, O

    1992-01-01

    Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a quiescent state in the adult. However, extensive proliferation can be induced in the adult liver by partial hepatectomy. In this study, we examined the regulation of ribosomal protein (rp) gene expression in the developing and regenerating rat liver. Our results indicate that the translation of rp mRNAs is selectively repressed by about 70% upon development from fetal to adult life, as illustrated by the decrease in ribosomal loading. In addition, the relative abundance of these mRNAs, like that of several other, but not all, housekeeping mRNAs, declines during development through a posttranscriptional mechanism. When liver cells commence growth following partial hepatectomy, translation of rp mRNAs is resumed to near-maximal capacity, as judged by their very efficient recruitment into polysomes. The concomitant increase in the abundance rp mRNAs under these circumstances is achieved by a posttranscriptional mechanism. The apparent fluctuations in the translation efficiency of rp mRNAs are accompanied by parallel changes in the expression of the genes encoding the initiation factors eIF-4E and eIF-4A. Our results indicate that selective translational control of rp mRNAs in mammals is not confined to manipulated cells in culture but constitutes an important regulatory mechanism operating in vivo in the course of liver development and regeneration. Images PMID:1373810

  4. Expression of cytochrome P450 regulators in cynomolgus macaque.

    PubMed

    Uno, Yasuhiro; Yamazaki, Hiroshi

    2017-09-11

    1. Cytochrome P450 (P450) regulators including nuclear receptors and transcription factors have not been fully investigated in cynomolgus macaques, an important species used in drug metabolism studies. In this study, we analyzed 17 P450 regulators by sequence and phylogenetic analysis, and tissue expression. 2. Gene and genome structures of 17 P450 regulators were similar to the human orthologs, and the deduced amino acid sequences showed high sequence identities (92-95%) and more closely clustered in a phylogenetic tree, with the human orthologs. 3. Many of the P450 regulator mRNAs were preferentially expressed in the liver, kidney, and/or jejunum. Among the P450 regulator mRNAs, PXR was most abundant in the liver and jejunum, and HNF4α in the kidney. In the liver, the expression of most P450 regulator mRNAs did not show significant differential expression (>2.5-fold) between cynomolgus macaques bred in Cambodia, China, and Indonesia, or rhesus macaques. 4. By correlation analysis, most of the P450 regulators were significantly (p < 0.05) correlated to other P450 regulators, and many of them were also significantly (p < 0.05) correlated with P450s. 5. These results suggest that 17 P450 regulators of cynomolgus macaques had similar molecular characteristics to the human orthologs.

  5. Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.

    PubMed Central

    Saber, M A; Zern, M A; Shafritz, D A

    1983-01-01

    We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated semiquantitatively by counting exposure of grains in autoradiographic emulsion placed over the cells. With this method of in situ hybridization, all hepatocytes appear to have significant, but highly variable, amounts of albumin mRNA. In addition, type I procollagen mRNA appears to be present at low abundance in hepatocytes. These results indicate that in situ hybridization can effectively demonstrate the presence of specific low- or high-abundance mRNAs in isolated well-differentiated eukaryotic cells. Images PMID:6575392

  6. The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

    PubMed Central

    Grönke, Sebastian; Stewart, James B.; Mourier, Arnaud; Ruzzenente, Benedetta; Kukat, Christian; Wibom, Rolf; Habermann, Bianca; Partridge, Linda; Larsson, Nils-Göran

    2011-01-01

    The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation. PMID:22022283

  7. Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs

    PubMed Central

    Diot, Cédric; Fournier, Guillaume; Dos Santos, Mélanie; Magnus, Julie; Komarova, Anastasia; van der Werf, Sylvie; Munier, Sandie; Naffakh, Nadia

    2016-01-01

    Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19. PMID:27653209

  8. The efficiency of 3'-end formation contributes to the relative levels of different histone mRNAs.

    PubMed Central

    Liu, T J; Levine, B J; Skoultchi, A I; Marzluff, W F

    1989-01-01

    Sequences at both the 5' and 3' ends of mouse histone genes contribute to the expression of individual genes. The 3' sequences required for high expression of the mouse H2a-614 gene are the same as the sequences required for 3'-end formation. When these sequences were substituted for the 3' end of the poorly expressed H2a-291 gene, expression of the H2a-291 gene was increased fivefold. A 65-nucleotide fragment containing the H2a-614 3' processing signal increased expression of the H2a-291 gene when it was placed in the proper orientation downstream of the H2a-291 3' end. The only mRNAs that accumulated from this gene ended at the H2a-291 3' end, which suggests that the transcript is sequentially processed. In an in vitro processing system, the different histone 3' ends showed different processing efficiencies, which correlated with their expression in cells. These results suggest that the efficiency of processing is important in determining the steady-state levels of individual mouse histone mRNAs. Images PMID:2796992

  9. Two classes of proteins and mRNAs in Lilium longiflorum L. indentified by human vitronectin probes.

    PubMed Central

    Wang, C S; Walling, L L; Gu, Y Q; Ware, C F; Lord, E M

    1994-01-01

    Vitronectin (VN) is a substrate adhesion molecule, an extracellular matrix glycoprotein that facilitates cell adhesion and cell movement in animals. We have reported the cross-reactivity of a 55-kD protein in plants with rabbit anti-human VN antibodies and the presence of VN-like sequences in plant genomes using a human VN cDNA probe. We have extended these studies by using human VN riboprobes to detect VN-like mRNAs in lily (Lilium longiflorum L.) and soybean. In both species, two mRNAs were detected. We have also identified a new cross-reactive protein (41 kD) using a different preparation of human VN antiserum. In lily roots five 41-kD isoforms were observed, whereas only three of these isoforms accumulated in leaves. Monospecific antibodies prepared against the plant proteins cross-reacted with the human VN protein and vice versa. We have purified the 41-kD protein using two-dimensional gel electrophoresis, and amino acid composition analysis indicates that it is similar in composition to human VN. PMID:7512737

  10. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

    PubMed

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

    Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  11. Translational efficiency of polycistronic mRNAs and their utilization to express heterologous genes in mammalian cells.

    PubMed Central

    Kaufman, R J; Murtha, P; Davies, M V

    1987-01-01

    The translation of polycistronic mRNAs in mammalian cells was studied. Transcription units, constructed to contain one, two or three open reading frames (ORFs), were introduced stably into Chinese hamster ovary cells and transiently into COS monkey cells. The analysis of mRNA levels and protein synthesis in these cells demonstrated that the mRNAs transcribed were translated to generate multiple proteins. The efficiency of translation was reduced approximately 40- to 300-fold by the insertion of an upstream ORF. The results support a modified 'scanning' model for translation initiation which allows for translation initiation at internal AUG codons. High-level expression of human granulocyte-macrophage colony stimulating factor was achieved utilizing a vector that contains a polycistronic transcription unit encoding an amplifiable dihydrofolate reductase marker gene in its 3' end. Thus, polycistronic expression vectors can be exploited to obtain high-level expression of foreign genes in mammalian cells. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3582359

  12. A 3'-coterminal nested set of independently transcribed mRNAs is generated during Berne virus replication.

    PubMed Central

    Snijder, E J; Horzinek, M C; Spaan, W J

    1990-01-01

    By using poly(A)-selected RNA from Berne virus (BEV)-infected embryonic mule skin cells as a template, cDNA was prepared and cloned in plasmid pUC9. Recombinants covering a contiguous sequence of about 10 kilobases were identified. Northern (RNA) blot hybridizations with various restriction fragments from these clones showed that the five BEV mRNAs formed a 3'-coterminal nested set. Sequence analysis revealed the presence of four complete open reading frames of 4743, 699, 426, and 480 nucleotides, with initiation codons coinciding with the 5' ends of BEV RNAs 2 through 5, respectively. By using primer extension analysis and oligonucleotide hybridizations, RNA 5 was found to be contiguous on the consensus sequence. The transcription of BEV mRNAs was studied by means of UV mapping. BEV RNAs 1, 2, and 3 were shown to be transcribed independently, which is also likely--although not rigorously proven--for RNAs 4 and 5. Upstream of the AUG codon of each open reading frame a conserved sequence pattern was observed which is postulated to function as a core promoter sequence in subgenomic RNA transcription. In the area surrounding the core promoter region of the two most abundant subgenomic BEV RNAs, a number of homologous sequence motifs were identified. Images PMID:2293666

  13. Evidence for tryptophan hydroxylase and hydroxy-indol-O-methyl-transferase mRNAs in human blood platelets.

    PubMed

    Champier, J; Claustrat, B; Besançon, R; Eymin, C; Killer, C; Jouvet, A; Chamba, G; Fèvre-Montange, M

    1997-01-01

    Human blood platelets were tested for the presence of mRNAs coding for tryptophan hydroxylase (TPOH) and hydroxy-indol-o-methyl-transferase (HIOMT). Total RNA was extracted from platelets (12.9 +/- 3.3 mg RNA/100 ml blood, mean +/- SEM of 6 preparations) and cDNA synthesized by reverse transcription using random hexamers, oligo-dT or TPOH- or HIOMT-specific primers, designed to amplify a 254 bp fragment for TPOH and a 301 bp fragment for HIOMT. Positive controls were performed using RNA extracted from human normal or tumoral pineal glands. The PCR products were analyzed by gel electrophoresis, transferred to a nylon membrane and hybridized with a 32P-labeled internal probe. When random hexamers, oligo-dT or specific primers were used for reverse transcription, amplification products of the predicted sizes were detectable following electrophoresis in the case of pineal glands and following transfer and hybridization in the case of platelets. These results show TPOH and HIOMT mRNAs to be present in human blood and support the hypothesis that serotonin and melatonin may be synthesized in blood and, more particularly, in platelets.

  14. The 5' ends of Thogoto virus (Orthomyxoviridae) mRNAs are homogeneous in both length and sequence.

    PubMed Central

    Albo, C; Martín, J; Portela, A

    1996-01-01

    Thogoto (THO) virus is a tick-borne member of the Orthomyxoviridae whose genome consists of six segments of linear, negative sense, single-stranded RNA. To gain insight into the mechanism by which viral mRNA transcripts are initiated, poly(A)+ RNA isolated from THO virus-infected cells was characterized by (i) primer extension experiments, (ii) immunoprecipitation studies with an anticap monoclonal antibody, (iii) direct sequencing analysis of the isolated RNA, and (iv) cloning and sequencing of individual mRNA molecules. The results indicated that THO virus mRNAs are capped and homogeneous in both length and sequence at their 5' end. These findings contrast with the situation found in all other segmented, negative sense or ambisense, single-stranded RNA viruses so far analyzed in which the 5' ends of viral mRNAs are heterogeneous in length and sequence. These results are discussed in terms of the mechanism used by THO virus to initiate mRNA synthesis. PMID:8971034

  15. The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs

    PubMed Central

    Thompson, Mary K; Rojas-Duran, Maria F; Gangaramani, Paritosh; Gilbert, Wendy V

    2016-01-01

    Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational ‘closed loop’ complex comprised of eIF4E, eIF4G, and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions. DOI: http://dx.doi.org/10.7554/eLife.11154.001 PMID:27117520

  16. Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs

    PubMed Central

    Boyne, James R; Jackson, Brian R; Taylor, Adam; Macnab, Stuart A; Whitehouse, Adrian

    2010-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts. PMID:20436455

  17. Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

    PubMed Central

    Balagopal, Vidya; Beemon, Karen L.

    2017-01-01

    All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

  18. Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

    SciTech Connect

    Mayman, B.A.; Nishioka, Y.

    1985-01-01

    The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed.

  19. Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.

    PubMed Central

    Akagi, H; Patton, D E; Miledi, R

    1989-01-01

    Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors. Images PMID:2479016

  20. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  1. Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP).

    PubMed

    Hecht, N B

    2000-06-01

    Functions ranging from RNA transport and translational regulation to DNA rearrangement and repair have been proposed for the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP). TB-RBP is primarily in the nuclei of male germ cells during meiosis and in the cytoplasm of male cells after metaphase I of meiosis. Based on its shift in subcellular locations as germ cells differentiate and its binding to microtubules and microfilaments, a model is presented proposing an involvement of TB-RBP in mRNA transport from nucleus to cytoplasm and in the sharing of mRNAs transcribed from the sex chromosomes by movement through intercellular bridges of germ cells.

  2. Expression of adenovirus-2 early region 4: assignment of the early region 4 polypeptides to their respective mRNAs, using in vitro translation.

    PubMed

    Tigges, M A; Raskas, H J

    1982-12-01

    Adenovirus-2 early region 4 (E4; map positions 91.3 to 99.1) encodes six 5' and 3' coterminal, differently spliced mRNAs, which are 2.5, 2.1, 1.8, 1.5, 1.2, and 0.8 kilobases (kb) long. Hybridization selection with five cloned viral DNA fragments that hybridize with subsets of E4 mRNAs was used to purify these six mRNAs and a previously unreported 3.0-kb mRNA from virus-infected cells. E4 mRNAs which were purified by hybridization selection with cloned EcoRI fragment C (map positions 89.7 to 100) were also fractionated by size. The purified mRNAs were then translated in rabbit reticulocyte or wheat germ lysate systems. The full complement of E4 mRNAs specified as many as 16 different polypeptides, with molecular weights ranging from 24,000 (24K) to 10K. The most abundant E4 mRNA, which was 2.1 kb long, specified an 11K polypeptide. The 1.5-kb mRNA, which differed from the 2.1-kb mRNA only by deletion of a second intron from the 3' untranslated region, also specified an 11K polypeptide. The second most abundant mRNA, which was 1.8 kb long, and the 1.2-kb mRNA, which had an intron deleted from the 3' untranslated region, specified a 15K polypeptide. This polypeptide was labeled more intensely with [5,6-(3)H]leucine than with [35S]methionine. The 3.0- and 2.5-kb mRNAs specified four polypeptides (24K, 22K, 19K, and 17K). Translation of E4 mRNAs with a mean size of 0.8 kb, which accumulated preferentially in the presence of cycloheximide, yielded at least 10 polypeptides that migrated in polyacrylamide gels with apparent molecular weights ranging from 21,800 to 10,000. On the basis of translation in wheat germ lysates and the distribution of polypeptides encoded by size-fractionated mRNAs, we concluded that the 0.8-kb mRNA size class includes a heterogeneous mixture of mRNAs which are probably formed as the result of utilization of alternate splice acceptor and donor sites during removal of the second intron. Our polypeptide assignments for the 2.1-, 1.8-, 1.5-, and 1

  3. Expression of transforming growth factor-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo.

    PubMed

    Jakowlew, S B; Ciment, G; Tuan, R S; Sporn, M B; Roberts, A B

    1994-01-01

    Specific cDNA probes and antibodies for chicken transforming growth factor (TGF)-beta 2 and beta 3 were used to study expression of TGF-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo. Expression of the mRNAs for both TGF-beta isoforms was detected by day 1.5 of incubation (Hamburger and Hamilton stage 10) by RNA Northern blot analysis and increased with developmental age. Expression of TGF-beta 2 and beta 3 mRNAs was detected in every embryonic tissue examined, with the level of expression of both isoforms being high in heart, brain and muscle and low in kidney and liver. Coordinate unidirectional upregulation of expression of TGF-beta 2 and beta 3 mRNAs occurred in most embryonic tissues with development except the heart, where the steady-state level of expression of TGF-beta 2 mRNA decreased with age, while that of TGF-beta 3 mRNA increased. In situ hybridization analysis detected TGF-beta 2 and beta 3 mRNAs as early as the definitive primitive streak stage (stage 4). During neurulation (stage 10), TGF-beta 2 and beta 3 mRNAs were detected in cells of all three germ layers; TGF-beta 3 mRNA was detected in neurectoderm as well. Following neurulation, TGF-beta 3 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome and dermomyotome at stage 16; expression of TGF-beta 2 mRNA was not as prominent as TGF-beta 3 mRNA in these structures. By stage 29, both TGF-beta 2 and beta 3 mRNAs were localized in several tissues including heart, lung, gizzard and feathers. Immunohistochemical staining analysis detected immunoreactive TGF-beta 2 and beta 3 proteins in all three germ layers of stage 4 embryos. Staining for TGF-beta 2 and beta 3 proteins was detected in several cell types and tissues in the early developing embryo frequently in the same locations as TGF-beta 2 and beta 3 mRNAs, with staining for TGF-beta 2 being less intense than TGF-beta 3. However, in some cases, localization of TGF-beta 2 and beta 3 proteins was

  4. Epstein-Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs.

    PubMed

    Juillard, Franceline; Bazot, Quentin; Mure, Fabrice; Tafforeau, Lionel; Macri, Christophe; Rabourdin-Combe, Chantal; Lotteau, Vincent; Manet, Evelyne; Gruffat, Henri

    2012-08-01

    The Epstein-Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human β-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the β-globin transcript suggesting that EB2 acts by stabilizing SRp20's labile interactions with the RNA.

  5. Epstein–Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs

    PubMed Central

    Juillard, Franceline; Bazot, Quentin; Mure, Fabrice; Tafforeau, Lionel; Macri, Christophe; Rabourdin-Combe, Chantal; Lotteau, Vincent; Manet, Evelyne; Gruffat, Henri

    2012-01-01

    The Epstein–Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human β-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the β-globin transcript suggesting that EB2 acts by stabilizing SRp20’s labile interactions with the RNA. PMID:22505578

  6. Cellular hybridization for BDNF, trkB, and NGF mRNAs and BDNF-immunoreactivity in rat forebrain after pilocarpine-induced status epilepticus.

    PubMed

    Schmidt-Kastner, R; Humpel, C; Wetmore, C; Olson, L

    1996-01-01

    The messenger RNAs (mRNAs) for the neurotrophins, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), are upregulated during epileptic seizure activity, as visualized by in situ hybridization techniques. Neurotrophins might be protective against excitotoxic cell stress, and the upregulation during seizures might provide such cell protection. In this study, a high dose of pilocarpine (300 mg/kg) was used to induce long-lasting, limbic motor status epilepticus and a selective pattern of brain damage. The regulation of BDNF, trkB, and NGF mRNA was studied by in situ hybridization at 1, 3, 6, and 24 h after induction of limbic motor status epilepticus. BDNF immunoreactivity was examined with an anti-peptide antibody and the neuropathological process studied in parallel. BDNF mRNA increased in hippocampus, neocortex, piriform cortex, striatum, and thalamus with a maximum at 3-6 h. Hybridization levels increased earlier in the resistant granule and CA1 cells as compared to the vulnerable CA3 neurons. BDNF immunoreactivity was elevated in dentate gyrus at 3-6 h. trkB mRNA increased in the entire hippocampus. NGF mRNA in hippocampus appeared in dentate gyrus at 3-6 h and declined in hilar neurons at 6-24 h. Cell damage was found in the CA3 area, entire basal cortex, and layers II/III of neocortex. Endogenous neurotrophins are upregulated during status epilepticus caused by pilocarpine, which is related to the coupling between neuronal excitation and trophic factor expression. This upregulation of neurotrophic factors may serve endogenous protective effects; however, the excessive levels of neuronal hyperexcitation resulting from pilocarpine seizures lead to cell damage which cannot be prevented by endogenous neurotrophins.

  7. A global profiling of uncapped mRNAs under cold stress reveals specific decay patterns and endonucleolytic cleavages in Brachypodium distachyon

    PubMed Central

    2013-01-01

    Background mRNA degradation is a critical factor in determining mRNA abundance and enables rapid adjustment of gene expression in response to environmental stress. The involvement of processing bodies in stress response suggests a role for decapping-mediated mRNA degradation. However, little is known about the role of mRNA degradation under stressful environmental conditions. Results Here, we perform a global study of uncapped mRNAs, via parallel analysis of RNA ends (PARE), under cold stress in Brachypodium distachyon. Enrichment analysis indicates that degradation products detected by PARE are mainly generated by the decapping pathway. Endonucleolytic cleavages are detected, uncovering another way of modulating gene expression. PARE and RNA-Seq analyses identify four types of mRNA decay patterns. Type II genes, for which light-harvesting processes are over-represented in gene ontology analyses, show unchanged transcript abundance and altered uncapped transcript abundance. Uncapping-mediated transcript stability of light harvesting-related genes changes significantly in response to cold stress, which may allow rapid adjustments in photosynthetic activity in response to cold stress. Transcript abundance and uncapped transcript abundance for type III genes changes in opposite directions in response to cold stress, indicating that uncapping-mediated mRNA degradation plays a role in regulating gene expression. Conclusion To our knowledge, this is the first global analysis of mRNA degradation under environmental stress conditions in Brachypodium distachyon. We uncover specific degradation and endonucleolytic cleavage patterns under cold stress, which will deepen our understanding of mRNA degradation under stressful environmental conditions, as well as the cold stress response mechanism in monocots. PMID:24000894

  8. Hypoglycemic Effects of Three Medicinal Plants in Experimental Diabetes: Inhibition of Rat Intestinal α-glucosidase and Enhanced Pancreatic Insulin and Cardiac Glut-4 mRNAs Expression.

    PubMed

    Moradabadi, Leila; Montasser Kouhsari, Shideh; Fehresti Sani, Mohammad

    2013-01-01

    Garlic (Allium sativum L., Alliaceae), Persian shallot (Allium ascalonicum L., Alliaceae ) and Sage (Salvia officinalis L., Lamiaceae) are believed to have hypoglycemic properties and have been used traditionally as antidiabetic herbal medicines in Iran. In this study, diabetes was induced by subcutaneous injection of alloxan monohydrate (100 mg kg(-1)) to male Wistar rats. Antidiabetic effects of methanolic extracts of the above mentioned three plants on alloxan-diabetic rats was investigated in comparison with the effects of antidiabetic drugs such as acarbose, glibenclamide and metformin by measuring postprandial blood glucose (PBG), oral glucose tolerance test (OGTT), inhibition of rat intestinal α-glucosidase enzymes activities and pancreatic Insulin and cardiac Glut-4 mRNAs expression. In short term period, hypoglycemic effects of A. sativum and A. ascalonicum showed significant reduction of PBG similar to glibenclamide (5 mg kg(-1) bw) while S. officinalis significantly reduced PBG similar to acarbose (20 mg kg(-1) bw). After 3 weeks of treatment by methanolic plant extracts, significant chronic decrease in the PBG was observed similar to metformin (100 mg kg(-1) bw). For OGTT, S. officinalis reduced PBG in a similar way as acarbose (20 mg kg(-1) bw). Intestinal sucrase and maltase activities were inhibited significantly by A. sativum, A. ascalonicum and S. officinalis. In addition, we observed increased expression of Insulin and Glut-4 genes in diabetic rats treated with these plants extracts. Up regulation of Insulin and Glut-4 genes expression and inhibition of α-glucosidaseactivities are the two mechanisms that play a considerable role in hypoglycemic action of garlic, shallot and sage.

  9. Hypoglycemic Effects of Three Medicinal Plants in Experimental Diabetes: Inhibition of Rat Intestinal α-glucosidase and Enhanced Pancreatic Insulin and Cardiac Glut-4 mRNAs Expression

    PubMed Central

    Moradabadi, Leila; Montasser Kouhsari, Shideh; Fehresti Sani, Mohammad

    2013-01-01

    Garlic (Allium sativum L., Alliaceae), Persian shallot (Allium ascalonicum L., Alliaceae ) and Sage (Salvia officinalis L., Lamiaceae) are believed to have hypoglycemic properties and have been used traditionally as antidiabetic herbal medicines in Iran. In this study, diabetes was induced by subcutaneous injection of alloxan monohydrate (100 mg kg−1) to male Wistar rats. Antidiabetic effects of methanolic extracts of the above mentioned three plants on alloxan-diabetic rats was investigated in comparison with the effects of antidiabetic drugs such as acarbose, glibenclamide and metformin by measuring postprandial blood glucose (PBG), oral glucose tolerance test (OGTT), inhibition of rat intestinal α-glucosidase enzymes activities and pancreatic Insulin and cardiac Glut-4 mRNAs expression. In short term period, hypoglycemic effects of A. sativum and A. ascalonicum showed significant reduction of PBG similar to glibenclamide (5 mg kg−1 bw) while S. officinalis significantly reduced PBG similar to acarbose (20 mg kg−1 bw). After 3 weeks of treatment by methanolic plant extracts, significant chronic decrease in the PBG was observed similar to metformin (100 mg kg−1 bw). For OGTT, S. officinalis reduced PBG in a similar way as acarbose (20 mg kg−1 bw). Intestinal sucrase and maltase activities were inhibited significantly by A. sativum, A. ascalonicum and S. officinalis. In addition, we observed increased expression of Insulin and Glut-4 genes in diabetic rats treated with these plants extracts. Up regulation of Insulin and Glut-4 genes expression and inhibition of α-glucosidaseactivities are the two mechanisms that play a considerable role in hypoglycemic action of garlic, shallot and sage. PMID:24250646

  10. Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes.

    PubMed

    Zhu, P; Liu, Q; Liu, S; Su, X; Feng, W; Lei, X; Liu, J; Cui, K; Huang, B; Shi, D

    2015-06-01

    In this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3-targeted mice were generated by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial bio-companies. The TALENs mutagenic non-homologous end-joining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK-293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN-mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN-mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3-independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3(-/+) ), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide-type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENs-based gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.

  11. A global profiling of uncapped mRNAs under cold stress reveals specific decay patterns and endonucleolytic cleavages in Brachypodium distachyon.

    PubMed

    Zhang, Jingyu; Mao, Zhiwei; Chong, Kang

    2013-08-30

    mRNA degradation is a critical factor in determining mRNA abundance and enables rapid adjustment of gene expression in response to environmental stress. The involvement of processing bodies in stress response suggests a role for decapping-mediated mRNA degradation. However, little is known about the role of mRNA degradation under stressful environmental conditions. Here, we perform a global study of uncapped mRNAs, via parallel analysis of RNA ends (PARE), under cold stress in Brachypodium distachyon. Enrichment analysis indicates that degradation products detected by PARE are mainly generated by the decapping pathway. Endonucleolytic cleavages are detected, uncovering another way of modulating gene expression. PARE and RNA-Seq analyses identify four types of mRNA decay patterns. Type II genes, for which light-harvesting processes are over-represented in gene ontology analyses, show unchanged transcript abundance and altered uncapped transcript abundance. Uncapping-mediated transcript stability of light harvesting-related genes changes significantly in response to cold stress, which may allow rapid adjustments in photosynthetic activity in response to cold stress. Transcript abundance and uncapped transcript abundance for type III genes changes in opposite directions in response to cold stress, indicating that uncapping-mediated mRNA degradation plays a role in regulating gene expression. To our knowledge, this is the first global analysis of mRNA degradation under environmental stress conditions in Brachypodium distachyon. We uncover specific degradation and endonucleolytic cleavage patterns under cold stress, which will deepen our understanding of mRNA degradation under stressful environmental conditions, as well as the cold stress response mechanism in monocots.

  12. Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinomas

    PubMed Central

    2011-01-01

    Background Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer. Findings PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas. Conclusion Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs. PMID:21504585

  13. Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas.

    PubMed

    Corbeil, Serge; Faury, Nicole; Segarra, Amélie; Renault, Tristan

    2015-01-01

    An in situ hybridization protocol for detecting mRNAs of ostreid herpesvirus type 1 (OsHV-1) which infects Pacific oysters, Crassostrea gigas, was developed. Three RNA probes were synthesized by cloning three partial OsHV-1 genes into plasmids using three specific primer pairs, and performing a transcription in the presence of digoxigenin dUTP. The RNA probes were able to detect the virus mRNAs in paraffin sections of experimentally infected oysters 26 h post-injection. The in situ hybridization showed that the OsHV-1 mRNAs were mainly present in connective tissues in gills, mantle, adductor muscle, digestive gland and gonads. DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also performed and results were compared with those obtained using RNA probes.

  14. Selective Changes of GABAA Channel Subunit mRNAs in the Hippocampus and Orbitofrontal Cortex but not in Prefrontal Cortex of Human Alcoholics

    PubMed Central

    Jin, Zhe; Bazov, Igor; Kononenko, Olga; Korpi, Esa R.; Bakalkin, Georgy; Birnir, Bryndis

    2012-01-01

    Alcohol dependence is a common chronic relapsing disorder. The development of alcohol dependence has been associated with changes in brain GABAA channel-mediated neurotransmission and plasticity. We have examined mRNA expression of the GABAA channel subunit genes in three brain regions in individuals with or without alcohol dependence using quantitative real-time PCR assay. The levels of selective GABAA channel subunit mRNAs were altered in specific brain regions in alcoholic subjects. Significant increase in the α1, α4, α5, β1, and γ1 subunit mRNAs in the hippocampal dentate gyrus region, and decrease in the β2 and δ subunit mRNAs in the orbitofrontal cortex were identified whereas no changes in the dorsolateral prefrontal cortex were detected. The data increase our understanding of the role of GABAA channels in the development of alcohol dependence. PMID:22319468

  15. Identification and developmental expression of mRNAs encoding putative insect cuticle hardening hormone, bursicon in the green shore crab Carcinus maenas.

    PubMed

    Wilcockson, David C; Webster, Simon G

    2008-03-01

    Bursicon is the ultimate hormone in insect ecdysis, which is involved in cuticle hardening. Here we show that mRNAs encoding the heterodimeric cystine knot protein bursicon (Burs alpha, beta), are present in crustaceans, suggesting ubiquity of this hormone in arthropods. We firstly report the cloning, sequencing of mRNAs encoding subunits from the water flea, Daphnia arenata and the CNS of the crab, Carcinus maenas, in comparison with insect bursicon subunits. Expression patterns of alpha and beta burs mRNAs were examined by in-situ hybridisation (ISH) and quantitative RT-PCR. In the thoracic ganglion, burs alpha and beta mRNAs were completely colocalised in neurones expressing crustacean cardioactive peptide (CCAP). However, in the brain and eyestalk, bursicon transcripts were never observed, despite a complex expression pattern of CCAP interneurones. Patterns of expression of burs alpha and beta mRNAs were constitutive during the moult cycle of adult crabs, in stark contrast to the situation in insects. Whilst copy numbers of burs beta transcripts closely matched those of CCAP, those of burs alpha mRNA were around 3-fold higher than burs beta. This pattern was apparent during embryogenesis, where bursicon transcripts were first observed at around 50% development-the same time as first expression of CCAP mRNA. Transcript ratios (burs alpha: beta) increased during development. Our studies have shown, for the first time, that bursicon mRNAs are expressed in identified neurones in the nervous system of crustaceans. These findings will now promote further investigation into the functions of bursicon during the moult cycle and development of crustaceans.

  16. Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs.

    PubMed Central

    Min, J; Zassenhaus, H P

    1993-01-01

    An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs. Images PMID:7686618

  17. Multiple Mobile mRNA Signals Regulate Tuber Development in Potato.

    PubMed

    Hannapel, David J; Banerjee, Anjan K

    2017-02-10

    Included among the many signals that traffic through the sieve element system are full-length mRNAs that function to respond to the environment and to regulate development. In potato, several mRNAs that encode transcription factors from the three-amino-loop-extension (TALE) superfamily move from leaves to roots and stolons via the phloem to control growth and signal the onset of tuber formation. This RNA transport is enhanced by short-day conditions and is facilitated by RNA-binding proteins from the polypyrimidine tract-binding family of proteins. Regulation of growth is mediated by three mobile mRNAs that arise from vasculature in the leaf. One mRNA, StBEL5, functions to activate growth, whereas two other, sequence-related StBEL's, StBEL11 and StBEL29, function antagonistically to repress StBEL5 target genes involved in promoting tuber development. This dynamic system utilizes closely-linked phloem-mobile mRNAs to control growth in developing potato tubers. In creating a complex signaling pathway, potato has evolved a long-distance transport system that regulates underground organ development through closely-associated, full-length mRNAs that function as either activators or repressors.

  18. Multiple Mobile mRNA Signals Regulate Tuber Development in Potato

    PubMed Central

    Hannapel, David J.; Banerjee, Anjan K.

    2017-01-01

    Included among the many signals that traffic through the sieve element system are full-length mRNAs that function to respond to the environment and to regulate development. In potato, several mRNAs that encode transcription factors from the three-amino-loop-extension (TALE) superfamily move from leaves to roots and stolons via the phloem to control growth and signal the onset of tuber formation. This RNA transport is enhanced by short-day conditions and is facilitated by RNA-binding proteins from the polypyrimidine tract-binding family of proteins. Regulation of growth is mediated by three mobile mRNAs that arise from vasculature in the leaf. One mRNA, StBEL5, functions to activate growth, whereas two other, sequence-related StBEL’s, StBEL11 and StBEL29, function antagonistically to repress StBEL5 target genes involved in promoting tuber development. This dynamic system utilizes closely-linked phloem-mobile mRNAs to control growth in developing potato tubers. In creating a complex signaling pathway, potato has evolved a long-distance transport system that regulates underground organ development through closely-associated, full-length mRNAs that function as either activators or repressors. PMID:28208608

  19. Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors

    PubMed Central

    Goparaju, Sravan Kumar; Kohda, Kazuhisa; Ibata, Keiji; Soma, Atsumi; Nakatake, Yukhi; Akiyama, Tomohiko; Wakabayashi, Shunichi; Matsushita, Misako; Sakota, Miki; Kimura, Hiromi; Yuzaki, Michisuke; Ko, Shigeru B. H.; Ko, Minoru S. H.

    2017-01-01

    Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs. PMID:28205555

  20. Molecular beacon-enabled purification of living cells by targeting cell type-specific mRNAs.

    PubMed

    Wile, Brian M; Ban, Kiwon; Yoon, Young-Sup; Bao, Gang

    2014-10-01

    Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

  1. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    PubMed Central

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  2. Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis

    PubMed Central

    Liu, Ming-Jung; Wu, Szu-Hsien; Chen, Ho-Ming; Wu, Shu-Hsing

    2012-01-01

    Environmental ‘light' has a vital role in regulating plant growth and development. Transcriptomic profiling has been widely used to examine how light regulates mRNA levels on a genome-wide scale, but the global role of translational regulation in the response to light is unknown. Through a transcriptomic comparison of steady-state and polysome-bound mRNAs, we reveal a clear impact of translational control on thousands of genes, in addition to transcriptomic changes, during photomorphogenesis. Genes encoding ribosomal protein are preferentially regulated at the translational level, which possibly contributes to the enhanced translation efficiency. We also reveal that mRNAs regulated at the translational level share characteristics of longer half-lives and shorter cDNA length, and that transcripts with a cis-element, TAGGGTTT, in their 5′ untranslated region have higher translatability. We report a previously neglected aspect of gene expression regulation during Arabidopsis photomorphogenesis. The identities and molecular signatures associated with mRNAs regulated at the translational level also offer new directions for mechanistic studies of light-triggered translational enhancement in Arabidopsis. PMID:22252389

  3. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs

    PubMed Central

    Vlasova-St. Louis, Irina; Bohjanen, Paul R.

    2016-01-01

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP. PMID:26821046

  4. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs.

    PubMed

    Vlasova-St Louis, Irina; Bohjanen, Paul R

    2016-01-25

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP.

  5. Endothelin: Visualization of mRNAs by in situ hybridization provides evidence for local action

    SciTech Connect

    MacCumber, M.W.; Ross, C.A.; Glaser, B.M.; Snyder, S.H. )

    1989-09-01

    Endothelin (ET) is a recently identified vasoactive peptide with three isoforms for which three genes have been cloned. The cellular sites of synthesis of this peptide have not yet been identified in vivo. Using Northern blot analysis, we have detected two forms of ET mRNA in rat tissues: a 3.7-kilobase form in the kidney, eye, and brain, a 2.5-kilobase form in the intestine, and both forms in the lung. We have localized these forms of ET mRNA in several rat tissues using in situ hybridization. In the 19-day rat fetus, ET mRNA is highest in the lung, intestine, and meninges. At high resolution, ET mRNA is localized in the lung to respiratory epithelial cells of bronchioles and apparently in blood vessels. In adult tissues, ET mRNA is present throughout the lung, in the renal medulla vasa recta, and in the iris of the eye. ET mRNA is synthesized in close proximity to ET binding sites in many organs (e.g., lung, kidney, intestine, and eye), suggesting a local action of this peptide. However, in other areas (e.g., heart and renal cortex), ET binding sites are present in the absence of ET mRNA, suggesting an action of ET from the bloodstream or from neurons. Northern blot analysis of ET mRNA in microvascular endothelial cells in culture indicates that ET is synthesized in small blood vessels and regulated similarly to its regulation in large vessels. Our results provide evidence that ET, like other regulatory peptides, may serve in several tissues as a neuromodulator or local hormone.

  6. A cellular mechanism for targeting newly synthesized mRNAs to synaptic sites on dendrites

    PubMed Central

    Steward, Oswald; Worley, Paul F.

    2001-01-01

    Long-lasting forms of activity-dependent synaptic plasticity involve molecular modifications that require gene expression. Here, we describe a cellular mechanism that mediates the targeting newly synthesized gene transcripts to individual synapses where they are locally translated. The features of this mechanism have been revealed through studies of the intracellular transport and synaptic targeting of the mRNA for a recently identified immediate early gene called activity-regulated cytoskeleton-associated protein Arc. Arc is strongly induced by patterns of synaptic activity that also induce long-term potentiation, and Arc mRNA is then rapidly delivered into dendrites after episodes of neuronal activation. The newly synthesized Arc mRNA localizes selectively at synapses that recently have been activated, and the encoded protein is assembled into the synaptic junctional complex. The dynamics of trafficking of Arc mRNA reveal key features of the mechanism through which synaptic activity can both induce gene expression and target particular mRNA transcripts to the active synapses. PMID:11416188

  7. G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

    PubMed

    Bidet, Katell; Dadlani, Dhivya; Garcia-Blanco, Mariano A

    2014-07-01

    Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

  8. G3BP1, G3BP2 and CAPRIN1 Are Required for Translation of Interferon Stimulated mRNAs and Are Targeted by a Dengue Virus Non-coding RNA

    PubMed Central

    Bidet, Katell; Dadlani, Dhivya; Garcia-Blanco, Mariano A.

    2014-01-01

    Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells. PMID:24992036

  9. The RNA recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is essential for plastid biogenesis and plant development.

    PubMed

    Teubner, Marlene; Fuß, Janina; Kühn, Kristina; Krause, Kirsten; Schmitz-Linneweber, Christian

    2017-02-01

    Chloroplast RNA metabolism depends on a multitude of nuclear-encoded RNA-binding proteins (RBPs). Most known chloroplast RBPs address specific RNA targets and RNA-processing functions. However, members of the small chloroplast ribonucleoprotein family (cpRNPs) play a global role in processing and stabilizing chloroplast RNAs. Here, we show that the cpRNP CP33A localizes to a distinct sub-chloroplastic domain and is essential for chloroplast development. The loss of CP33A yields albino seedlings that exhibit aberrant leaf development and can only survive in the presence of an external carbon source. Genome-wide RNA association studies demonstrate that CP33A associates with all chloroplast mRNAs. For a given transcript, quantification of CP33A-bound versus free RNAs demonstrates that CP33A associates with the majority of most mRNAs analyzed. Our results further show that CP33A is required for the accumulation of a number of tested mRNAs, and is particularly relevant for unspliced and unprocessed precursor mRNAs. Finally, CP33A fails to associate with polysomes or to strongly co-precipitate with ribosomal RNA, suggesting that it defines a ribodomain that is separate from the chloroplast translation machinery. Collectively, these findings suggest that CP33A contributes to globally essential RNA processes in the chloroplasts of higher plants.

  10. Red5 and three nuclear pore components are essential for efficient suppression of specific mRNAs during vegetative growth of fission yeast.

    PubMed

    Sugiyama, Tomoyasu; Wanatabe, Nobuyoshi; Kitahata, Eri; Tani, Tokio; Sugioka-Sugiyama, Rie

    2013-07-01

    Zinc-finger domains are found in many nucleic acid-binding proteins in both prokaryotes and eukaryotes. Proteins carrying zinc-finger domains have important roles in various nuclear transactions, including transcription, mRNA processing and mRNA export; however, for many individual zinc-finger proteins in eukaryotes, the exact function of the protein is not fully understood. Here, we report that Red5 is involved in efficient suppression of specific mRNAs during vegetative growth of Schizosaccharomyces pombe. Red5, which contains five C3H1-type zinc-finger domains, localizes to the nucleus where it forms discrete dots. A red5 point mutation, red5-2, results in the upregulation of specific meiotic mRNAs in vegetative mutant red5-2 cells; northern blot data indicated that these meiotic mRNAs in red5-2 cells have elongated poly(A) tails. RNA-fluorescence in situ hybridization results demonstrate that poly(A)(+) RNA species accumulate in the nucleolar regions of red5-deficient cells. Moreover, Red5 genetically interacts with several mRNA export factors. Unexpectedly, three components of the nuclear pore complex also suppress a specific set of meiotic mRNAs. These results indicate that Red5 function is important to meiotic mRNA degradation; they also suggest possible connections among selective mRNA decay, mRNA export and the nuclear pore complex in vegetative fission yeast.

  11. The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei

    PubMed Central

    Guo, Xuemin; Ernst, Nancy Lewis; Carnes, Jason; Stuart, Kenneth D.

    2010-01-01

    Most mitochondrial mRNAs in trypanosomes undergo uridine insertion/deletion editing that is catalyzed by ∼20S editosomes. The editosome component KREPA3 is essential for editosome structural integrity and its two zinc finger (ZF) motifs are essential for editing in vivo but not in vitro. KREPA3 function was further explored by examining the consequence of mutation of its N- and C- terminal ZFs (ZF1 and ZF2, respectively). Exclusively expressed myc-tagged KREPA3 with ZF2 mutation resulted in lower KREPA3 abundance and a relative increase in KREPA2 and KREL1 proteins. Detailed analysis of edited RNA products revealed the accumulation of partially edited mRNAs with less insertion editing compared to the partially edited mRNAs found in the cells with wild type KREPA3 expression. Mutation of ZF1 in TAP-tagged KREPA3 also resulted in accumulation of partially edited mRNAs that were shorter and only edited in the 3′-terminal editing region. Mutation of both ZFs essentially eliminated partially edited mRNA. The mutations did not affect gRNA abundance. These data indicate that both ZFs are essential for the progression of editing and perhaps its accuracy, which suggests that KREPA3 plays roles in the editing process via its ZFs interaction with editosome proteins and/or RNA substrates. PMID:20111718

  12. eIF4AIII enhances translation of nuclear cap-binding complex-bound mRNAs by promoting disruption of secondary structures in 5'UTR.

    PubMed

    Choe, Junho; Ryu, Incheol; Park, Ok Hyun; Park, Joori; Cho, Hana; Yoo, Jin Seon; Chi, Sung Wook; Kim, Min Kyung; Song, Hyun Kyu; Kim, Yoon Ki

    2014-10-28

    It has long been considered that intron-containing (spliced) mRNAs are translationally more active than intronless mRNAs (identical mRNA not produced by splicing). The splicing-dependent translational enhancement is mediated, in part, by the exon junction complex (EJC). Nonetheless, the molecular mechanism by which each EJC component contributes to the translational enhancement remains unclear. Here, we demonstrate the previously unappreciated role of eukaryotic translation initiation factor 4AIII (eIF4AIII), a component of EJC, in the translation of mRNAs bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein 80 (CBP80) and CBP20. eIF4AIII is recruited to the 5'-end of mRNAs bound by the CBC by direct interaction with the CBC-dependent translation initiation factor (CTIF); this recruitment of eIF4AIII is independent of the presence of introns (deposited EJCs after splicing). Polysome fractionation, tethering experiments, and in vitro reconstitution experiments using recombinant proteins show that eIF4AIII promotes efficient unwinding of secondary structures in 5'UTR, and consequently enhances CBC-dependent translation in vivo and in vitro. Therefore, our data provide evidence that eIF4AIII is a specific translation initiation factor for CBC-dependent translation.

  13. Regulation of Transcript Elongation

    PubMed Central

    Belogurov, Georgiy A.; Artsimovitch, Irina

    2015-01-01

    Bacteria lack subcellular compartments and harbor a single RNA polymerase that synthesizes both structural and protein-coding RNAs, which are cotranscriptionally processed by distinct pathways. Nascent rRNAs fold into elaborate secondary structures and associate with ribosomal proteins, whereas nascent mRNAs are translated by ribosomes. During elongation, nucleic acid signals and regulatory proteins modulate concurrent RNA-processing events, instruct RNA polymerase where to pause and terminate transcription, or act as roadblocks to the moving enzyme. Communications among complexes that carry out transcription, translation, repair, and other cellular processes ensure timely execution of the gene expression program and survival under conditions of stress. This network is maintained by auxiliary proteins that act as bridges between RNA polymerase, ribosome, and repair enzymes, blurring boundaries between separate information-processing steps and making assignments of unique regulatory functions meaningless. Understanding the regulation of transcript elongation thus requires genome-wide approaches, which confirm known and reveal new regulatory connections. PMID:26132790

  14. The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation.

    PubMed

    Dauber, Bianca; Poon, David; Dos Santos, Theodore; Duguay, Brett A; Mehta, Ninad; Saffran, Holly A; Smiley, James R

    2016-07-01

    The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects

  15. The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation

    PubMed Central

    Dauber, Bianca; Poon, David; dos Santos, Theodore; Duguay, Brett A.; Mehta, Ninad; Saffran, Holly A.

    2016-01-01

    ABSTRACT The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. IMPORTANCE The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least

  16. Expression of Alternatively Spliced Human T-Cell Leukemia Virus Type 1 mRNAs Is Influenced by Mitosis and by a Novel cis-Acting Regulatory Sequence

    PubMed Central

    Cavallari, Ilaria; Rende, Francesca; Bona, Marion K.; Sztuba-Solinska, Joanna; Silic-Benussi, Micol; Tognon, Martina; Franchini, Genoveffa; D'Agostino, Donna M.

    2015-01-01

    ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex–wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system. IMPORTANCE HTLV-1 is a complex retrovirus that causes two distinct pathologies termed adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy in about 5% of infected individuals. Expression of the virus depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of virus

  17. Expression of Alternatively Spliced Human T-Cell Leukemia Virus Type 1 mRNAs Is Influenced by Mitosis and by a Novel cis-Acting Regulatory Sequence.

    PubMed

    Cavallari, Ilaria; Rende, Francesca; Bona, Marion K; Sztuba-Solinska, Joanna; Silic-Benussi, Micol; Tognon, Martina; LeGrice, Stuart F J; Franchini, Genoveffa; D'Agostino, Donna M; Ciminale, Vincenzo

    2015-11-18

    Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system. HTLV-1 is a complex retrovirus that causes two distinct pathologies termed adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy in about 5% of infected individuals. Expression of the virus depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of virus expression. The findings reported

  18. Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia.

    PubMed

    Mohamed, J Shaik; Benninghoff, Abby D; Holt, G Joan; Khan, Izhar A

    2007-02-01

    The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups.

  19. Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein

    PubMed Central

    2014-01-01

    Background Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. Results To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Conclusions Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism. PMID:24393533

  20. Circadian control of mRNA polyadenylation dynamics regulates rhythmic protein expression

    PubMed Central

    Kojima, Shihoko; Sher-Chen, Elaine L.; Green, Carla B.

    2012-01-01

    Poly(A) tails are 3′ modifications of eukaryotic mRNAs that are important in the control of translation and mRNA stability. We identified hundreds of mouse liver mRNAs that exhibit robust circadian rhythms in the length of their poly(A) tails. Approximately 80% of these are primarily the result of nuclear adenylation coupled with rhythmic transcription. However, unique decay kinetics distinguish these mRNAs from other mRNAs that are transcribed rhythmically but do not exhibit poly(A) tail rhythms. The remaining 20% are uncoupled from transcription and exhibit poly(A) tail rhythms even though the steady-state mRNA levels are not rhythmic. These are under the control of rhythmic cytoplasmic polyadenylation, regulated at least in some cases by cytoplasmic polyadenylation element-binding proteins (CPEBs). Importantly, we found that the rhythmicity in poly(A) tail length is closely correlated with rhythmic protein expression, with a several-hour delay between the time of longest tail and the time of highest protein level. Our study demonstrates that the circadian clock regulates the dynamic polyadenylation status of mRNAs, which can result in rhythmic protein expression independent of the steady-state levels of the message. PMID:23249735

  1. Dynamically regulated miRNA-mRNA networks revealed by exercise

    PubMed Central

    2013-01-01

    Background MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. Results Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. Conclusions We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation. PMID:24219008

  2. Dynamically regulated miRNA-mRNA networks revealed by exercise.

    PubMed

    Tonevitsky, Alexander G; Maltseva, Diana V; Abbasi, Asghar; Samatov, Timur R; Sakharov, Dmitry A; Shkurnikov, Maxim U; Lebedev, Alexey E; Galatenko, Vladimir V; Grigoriev, Anatoly I; Northoff, Hinnak

    2013-06-07

    MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.

  3. mRNAs coding for neurotransmitter receptors and voltage-gated sodium channels in the adult rabbit visual cortex after monocular deafferentiation

    PubMed Central

    Nguyen, Quoc-Thang; Matute, Carlos; Miledi, Ricardo

    1998-01-01

    It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-d-aspartate (NMDA), γ-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system. PMID:9501250

  4. Virus-induced translational arrest through 4EBP1/2-dependent decay of 5′-TOP mRNAs restricts viral infection

    PubMed Central

    Hopkins, Kaycie C.; Tartell, Michael A.; Herrmann, Christin; Hackett, Brent A.; Taschuk, Frances; Panda, Debasis; Menghani, Sanjay V.; Sabin, Leah R.; Cherry, Sara

    2015-01-01

    The mosquito-transmitted bunyavirus, Rift Valley fever virus (RVFV), is a highly successful pathogen for which there are no vaccines or therapeutics. Translational arrest is a common antiviral strategy used by hosts. In response, RVFV inhibits two well-known antiviral pathways that attenuate translation during infection, PKR and type I IFN signaling. Despite this, translational arrest occurs during RVFV infection by unknown mechanisms. Here, we find that RVFV infection triggers the decay of core translation machinery mRNAs that possess a 5′-terminal oligopyrimidine (5′-TOP) motif in their 5′-UTR, including mRNAs encoding ribosomal proteins, which leads to a decrease in overall ribosomal protein levels. We find that the RNA decapping enzyme NUDT16 selectively degrades 5′-TOP mRNAs during RVFV infection and this decay is triggered in response to mTOR attenuation via the translational repressor 4EBP1/2 axis. Translational arrest of 5′-TOPs via 4EBP1/2 restricts RVFV replication, and this increased RNA decay results in the loss of visible RNA granules, including P bodies and stress granules. Because RVFV cap-snatches in RNA granules, the increased level of 5′-TOP mRNAs in this compartment leads to snatching of these targets, which are translationally suppressed during infection. Therefore, translation of RVFV mRNAs is compromised by multiple mechanisms during infection. Together, these data present a previously unknown mechanism for translational shutdown in response to viral infection and identify mTOR attenuation as a potential therapeutic avenue against bunyaviral infection. PMID:26038567

  5. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum

    PubMed Central

    Jagannathan, Sujatha; Reid, David W.; Cox, Amanda H.

    2014-01-01

    The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER. PMID:25142066

  6. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum.

    PubMed

    Jagannathan, Sujatha; Reid, David W; Cox, Amanda H; Nicchitta, Christopher V

    2014-10-01

    The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER. © 2014 Jagannathan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. Distribution of albumin and alpha-fetoprotein mRNAs in normal, hyperplastic, and preneoplastic rat liver.

    PubMed Central

    Alpini, G.; Aragona, E.; Dabeva, M.; Salvi, R.; Shafritz, D. A.; Tavoloni, N.

    1992-01-01

    The nature of bile duct-like (oval) cells proliferating during chemical hepatocarcinogenesis has been controversial. To investigate this issue further, the authors compared the hepatic distribution of albumin (ALB) and alpha-fetoprotein (AFP) mRNAs in rats in which oval cell proliferation was induced by feeding a choline-devoid diet containing 0.1% ethionine (CDE, a hepatocarcinogenic diet) with that in normal rats and in rats in which biliary epithelial cell hyperplasia was induced by either bile duct ligation or feeding alpha-naphthylisothiocyanate (ANIT). Northern blot analysis in parenchymal and nonparenchymal liver cells isolated from these animals demonstrated that ALB mRNA was present in the hepatocytes of both control and experimental animals, whereas this transcript was detected in nonparenchymal epithelial cells only in CDE-fed rats. Alpha-fetoprotein mRNA was not seen in either parenchymal or nonparenchymal cells isolated from normal or hyperplastic livers induced by bile duct ligation or ANIT feeding. In CDE-fed rats, however, both parenchymal and nonparenchymal cell populations displayed AFP message. In situ hybridization directly demonstrated nonparenchymal cell expression of both ALB and AFP transcripts in CDE-fed rats. Most surprisingly, ALB and AFP mRNAs were also detected by in situ hybridization in occasional nonparenchymal cells located in portal tracts near the limiting plate in normal liver, as well as under conditions associated with bile duct hyperplasia. Immunohistochemical studies of intermediate filament proteins, cytokeratin 19 (a marker of glandular epithelia), vimentin (a marker of mesenchymal lineage), and desmin (a marker of muscle cell differentiation) demonstrated that oval cells, as well as normal and hyperplastic bile duct cells, were positive for cytokeratin 19 and negative for both vimentin and desmin. Cytokeratin-positive oval cells formed duct profiles and were connected to preexisting ductules and ducts. These results are

  8. Translational Regulation in Nutrigenomics12

    PubMed Central

    Liu, Botao; Qian, Shu-Bing

    2011-01-01

    The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health. PMID:22332093

  9. Time-Dependent Expression Profiles of microRNAs and mRNAs in Rat Milk Whey

    PubMed Central

    Izumi, Hirohisa; Kosaka, Nobuyoshi; Shimizu, Takashi; Sekine, Kazunori; Ochiya, Takahiro; Takase, Mitsunori

    2014-01-01

    Functional RNAs, such as microRNA (miRNA) and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2) was markedly higher than that in mature milk whey (days 9 and 16). Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes) to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats. PMID:24533154

  10. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs

    PubMed Central

    Martínez de Alba, Angel Emilio; Moreno, Ana Beatriz; Gabriel, Marc; Mallory, Allison C.; Christ, Aurélie; Bounon, Rémi; Balzergue, Sandrine; Aubourg, Sebastien; Gautheret, Daniel; Crespi, Martin D.; Vaucheret, Hervé; Maizel, Alexis

    2015-01-01

    Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5′ cap structure and their subsequent 5′ to 3′ degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs. PMID:25694514

  11. Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns

    PubMed Central

    Wang, Baomin; Rekosh, David

    2015-01-01

    Intron retention is one of the least studied forms of alternative splicing. Through the use of retrovirus and other model systems, it was established many years ago that mRNAs with retained introns are subject to restriction both at the level of nucleocytoplasmic export and cytoplasmic expression. It was also demonstrated that specific cis-acting elements in the mRNA could serve to bypass these restrictions. Here we show that one of these elements, the constitutive transport element (CTE), first identified in the retrovirus MPMV and subsequently in the human NXF1 gene, is a highly conserved element. Using GERP analysis, CTEs with strong primary sequence homology, predicted to display identical secondary structure, were identified in NXF genes from >30 mammalian species. CTEs were also identified in the predicted NXF1 genes of zebrafish and coelacanths. The CTE from the zebrafish NXF1 was shown to function efficiently to achieve expression of mRNA with a retained intron in human cells in conjunction with zebrafish Nxf1 and cofactor Nxt proteins. This demonstrates that all essential functional components for expression of mRNA with retained introns have been conserved from fish to man. PMID:25605961

  12. Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.

    PubMed

    Trask, Heidi W; Cowper-Sal-lari, Richard; Sartor, Maureen A; Gui, Jiang; Heath, Catherine V; Renuka, Janhavi; Higgins, Azara-Jane; Andrews, Peter; Korc, Murray; Moore, Jason H; Tomlinson, Craig R

    2009-10-01

    With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.

  13. A 40 kd protein binds specifically to the 5'-untranslated regions of yeast mitochondrial mRNAs.

    PubMed Central

    Papadopoulou, B; Dekker, P; Blom, J; Grivell, L A

    1990-01-01

    Using a gel mobility shift assay we show that a 40 kd protein (p40), present in extracts of yeast mitochondria, binds specifically to the 5'-untranslated leader of cytochrome c oxidase subunit II mRNA. Binding of p40 to coxII RNA protects an 8-10 nucleotide segment from diethylpyocarbonate modification, indicating that the protein interacts with only a restricted region of the 5'-leader. This segment is located at position -12 with respect to the initiation AUG. Deletion of 10 nucleotides encompassing this site completely abolishes protein binding. Nevertheless, Bal31 deletion analysis within the coxII leader shows that a major part of the leader is essential for p40 binding, suggesting that binding of the protein is also dependent on secondary structural features. p40 binds to other mitochondrial leader mRNAs including those for coxI, coxIII and cyt b. p40 is present in a cytoplasmic (rho0) petite mutant lacking mitochondrial protein synthesis. It is therefore presumably nuclear encoded. The possible biological function of the protein is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. Fig. 9. PMID:1701144

  14. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    SciTech Connect

    Keryer-Bibens, Cecile; Legagneux, Vincent; Namanda-Vanderbeken, Allen; Cosson, Bertrand; Paillard, Luc; Poncet, Didier; Osborne, H. Beverley

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  15. Impact of Dietary Interventions on Noncoding RNA Networks and mRNAs Encoding Chromatin-Related Factors.

    PubMed

    Green, Christopher D; Huang, Yi; Dou, Xiaoyang; Yang, Liu; Liu, Yong; Han, Jing-Dong J

    2017-03-21

    Dietary interventions dramatically affect metabolic disease and lifespan in various aging models. Here, we profiled liver microRNA (miRNA), coding, and long non-coding RNA (lncRNA) expression by high-throughput deep sequencing in mice across multiple energy intake and expenditure interventions. Strikingly, three dietary intervention network design patterns were uncovered: (1) lifespan-extending interventions largely repressed the expression of miRNAs, lncRNAs, and transposable elements; (2) protein-coding mRNAs with expression positively correlated with long lifespan are highly targeted by miRNAs; and (3) miRNA-targeting interactions mainly target chromatin-related functions. We experimentally validated miR-34a, miR-107, and miR-212-3p targeting of the chromatin remodeler Chd1 and further demonstrate that Chd1 knockdown mimics high-fat diet and aging-induced gene expression changes and activation of transposons. Our findings demonstrate lifespan-extending diets repress miRNA-chromatin remodeler interactions and safeguard against deregulated transcription induced by aging and lifespan shortening diets, events linked by microRNA, chromatin, and ncRNA crosstalk.

  16. Effects of pergolide mesilate on metallothionein mRNAs expression in a mouse model for Parkinson disease.

    PubMed

    Ono, Shin-ichi; Hirai, Kohji; Tokuda, Ei-ichi

    2009-10-01

    Dopamine agonists have neuroprotective properties in addition to their original pharmacologic function. We examined the effects of pergolide mesilate (PM) on the levels of metallothionein mRNA expression and lipid peroxidation in the corpus striata of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonian mice. Mice were administered normal saline (vehicle as a control), PM, or MPTP. A consecutive 7-d administration of MPTP via a gastric tube at a dose of 30 mg/kg significantly decreased metallothionein (MT)-I mRNA expression but did not influence MT-III mRNA expression. Lipid peroxidation, measured as the production of malondialdehyde reactive substances, did not increase after MPTP treatment. Although PM administration alone did not effect MT-I expression, an additional consecutive 7-d administration of PM (30 mug/kg) following MPTP treatment recovered the decreased MT-I level and increased MT-III expression. Lipid peroxidation was significantly suppressed. These results suggest that PM exerts an antioxidative property through the induction of MT-I and MT-III mRNAs simultaneously in response to cellular and/or tissue injury.

  17. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs.

    PubMed

    Martínez de Alba, Angel Emilio; Moreno, Ana Beatriz; Gabriel, Marc; Mallory, Allison C; Christ, Aurélie; Bounon, Rémi; Balzergue, Sandrine; Aubourg, Sebastien; Gautheret, Daniel; Crespi, Martin D; Vaucheret, Hervé; Maizel, Alexis

    2015-03-11

    Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5' to 3' degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Mesangial cells express PDGF (platelet-derived growth factor) mRNAs and proliferate in response to PDGF

    SciTech Connect

    Shultz, P.J.; DiCorleto, P.E.; Silver, B.J.; Abboud, H.E. Research Institute of the Cleveland Clinic Foundation, OH )

    1988-10-01

    Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin and is released and/or synthesized by platelets, macrophages, endothelial cells and rat mesangial cells. In the present investigation, the authors found that human glomerular mesangial cells in culture release a PDGF-like protein which competes for {sup 125}I-PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. The competing and to a lesser extent the mitogenic activities present in the conditioned medium are partially recognized by an anti-PDGF antibody. Northern blot analysis of poly(A){sup +} RNA from human mesangial cells demonstrates the expression of both PDGF A- and B-chain mRNAs. PDGF also binds to mesangial cells in a specific manner and stimulates DNA synthesis and cell proliferation. These data suggest that a PDGF-like protein secreted by mesangial cells or released from platelets, monocytes, or endothelial cells during glomerular inflammation may function as an autocrine or a paracrine growth factor for these cells. The biological role of PDGF in mediating proliferative and other inflammatory events in the glomerulus remains to be identified.

  19. Differential cellular expression of galectin family mRNAs in the epithelial cells of the mouse digestive tract.

    PubMed

    Nio, Junko; Kon, Yasuhiro; Iwanaga, Toshihiko

    2005-11-01

    Galectin is an animal lectin that recognizes beta-galactosides of glycoconjugates and is abundant in the gut. This study revealed the cellular expression of galectin subtypes throughout the mouse digestive tract by in situ hybridization. Signals for five subtypes (galectin-2, -3, -4/6, and -7) were detected exclusively in the epithelia. In the glandular stomach, galectin-2 and -4/6 were predominantly expressed from gastric pits to neck of gastric glands, where mucous cells were the main cellular sources. The small intestine exhibited intense, maturation-associated expressions of galectin-2, -3, and -4/6 mRNAs. Galectin-2 was intensely expressed from crypts to the base of villi, whereas transcripts of galectin-3 gathered at villous tips. Signals for galectin-4/6 were most intense at the lower half of villi. Galectin-2 was also expressed in goblet cells of the small intestine but not in those of the large intestine. In the large intestine, galectin-4/6 predominated, and the upper half of crypts simultaneously contained transcripts of galectin-3. Stratified epithelium from the lip to forestomach and anus intensely expressed galectin-7 with weak expressions of galectin-3. Because galectins in the digestive tract may be multi-functional, information on their cell/stage-specific expression contributes to a better understanding of the functions and pathological involvements of galectins.

  20. The oxidative stress response in yeast cells involves changes in the stability of Aft1 regulon mRNAs.

    PubMed

    Castells-Roca, Laia; Mühlenhoff, Ulrich; Lill, Roland; Herrero, Enrique; Bellí, Gemma

    2011-07-01

    Saccharomyces cerevisiae can import iron through a high-affinity system consisting of the Ftr1/Fet3-mediated reductive pathway and the siderophore-mediated non-reductive one. Expression of components of the high-affinity system is controlled by the Aft1 transcriptional factor. In this study we show that, upon oxidative stress, Aft1 is transitorily internalized into the nucleus, followed by transcription activation of components of its regulon. In these conditions, the mRNA levels of the genes of the non-reductive pathway become increased, while those of FTR1 and FET3 remain low because of destabilization of the mRNAs. Consequently, the respective protein levels also remain low. Such mRNA destabilization is mediated by the general 5'-3' mRNA decay pathway and is independent of the RNA binding protein Cth2. Yeast cells are hypersensitive to peroxides in growth conditions where only the high-affinity reductive pathway is functional for iron assimilation. On the contrary, peroxide does not affect growth when iron uptake occurs exclusively through the non-reductive pathway. This reinforces the idea that upon oxidative stress S. cerevisiae cells redirect iron assimilation through the non-reductive pathway to minimize oxidative damage by the ferrous ions, which are formed during iron import through the Ftr1/Fet3 complexes.

  1. Aging and infection reduce expression of specific brain-derived neurotrophic factor mRNAs in hippocampus.

    PubMed

    Chapman, Timothy R; Barrientos, Ruth M; Ahrendsen, Jared T; Hoover, Jennifer M; Maier, Steven F; Patterson, Susan L

    2012-04-01

    Aging increases the likelihood of cognitive decline after negative life events such as infection or injury. We have modeled this increased vulnerability in aged (24-month-old), but otherwise unimpaired F344xBN rats. In these animals, but not in younger (3-month-old) counterparts, a single intraperitoneal injection of E. coli leads to specific deficits in long-term memory and long-lasting synaptic plasticity in hippocampal area CA1-processes strongly dependent on brain-derived neurotrophic factor (BDNF). Here we have investigated the effects of age and infection on basal and fear-conditioning-stimulated expression of Bdnf in hippocampus. We performed in situ hybridization with 6 probes recognizing: total (pan-)BDNF mRNA, the 4 predominant 5' exon-specific transcripts (I, II, IV, and VI), and BDNF mRNAs with a long 3' untranslated region (3' UTR). In CA1, aging reduced basal levels and fear-conditioning-induced expression of total BDNF mRNA, exon IV-specific transcripts, and transcripts with long 3' UTRs; effects of infection were similar and sometimes compounded the effects of aging. In CA3, aging reduced all of the transcripts to some degree; infection had no effect. Effects in dentate were minimal. Northern blot analysis confirmed an aging-associated loss of total BDNF mRNA in areas CA1 and CA3, and revealed a parallel, preferential loss of BDNF mRNA transcripts with long 3' UTRs.

  2. Multiple binding of repressed mRNAs by the P-body protein Rck/p54

    PubMed Central

    Ernoult-Lange, Michèle; Baconnais, Sonia; Harper, Maryannick; Minshall, Nicola; Souquere, Sylvie; Boudier, Thomas; Bénard, Marianne; Andrey, Philippe; Pierron, Gérard; Kress, Michel; Standart, Nancy; le Cam, Eric; Weil, Dominique

    2012-01-01

    Translational repression is achieved by protein complexes that typically bind 3′ UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5′ extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation. PMID:22836354

  3. SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

    SciTech Connect

    Escudero-Paunetto, Laurimar; Li Ling; Hernandez, Felicia P.; Sandri-Goldin, Rozanne M.

    2010-06-05

    Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.

  4. Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA

    PubMed Central

    Schuessler, Dorothée L; Cortes, Teresa; Fivian-Hughes, Amanda S; Lougheed, Kathryn E A; Harvey, Evelyn; Buxton, Roger S; Davis, Elaine O; Young, Douglas B

    2013-01-01

    In Mycobacterium tuberculosis, the genes Rv1954A–Rv1957 form an operon that includes Rv1955 and Rv1956 which encode the HigB toxin and the HigA antitoxin respectively. We are interested in the role and regulation of this operon, since toxin–antitoxin systems have been suggested to play a part in the formation of persister cells in mycobacteria. To investigate the function of the higBA locus, effects of toxin expression on mycobacterial growth and transcript levels were assessed in M. tuberculosis H37Rv wild type and in an operon deletion background. We show that expression of HigB toxin in the absence of HigA antitoxin arrests growth and causes cell death in M. tuberculosis. We demonstrate HigB expression to reduce the abundance of IdeR and Zur regulated mRNAs and to cleave tmRNA in M. tuberculosis, Escherichia coli and Mycobacterium smegmatis. This study provides the first identification of possible target transcripts of HigB in M. tuberculosis. PMID:23927792

  5. RNA splicing regulates the temporal order of TNF-induced gene expression.

    PubMed

    Hao, Shengli; Baltimore, David

    2013-07-16

    When cells are induced to express inflammatory genes by treatment with TNF, the mRNAs for the induced genes appear in three distinct waves, defining gene groups I, II, and III, or early, intermediate, and late genes. To examine the basis for these different kinetic classes, we have developed a PCR-based procedure to distinguish pre-mRNAs from mRNAs. It shows that the three groups initiate transcription virtually simultaneously but that delays in splicing characterize groups II and III. We also examined the elongation times, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production.

  6. Expression of mRNAs encoding receptors that mediate stress signals in gonadotropin-releasing hormone neurons of the mouse.

    PubMed

    Jasoni, Christine L; Todman, Martin G; Han, Seong-Kyu; Herbison, Allan E

    2005-01-01

    Neurons that synthesize and secrete gonadotropin-releasing hormone (GnRH) represent the neural control point for fertility modulation in vertebrates. As such GnRH neurons are ideally situated to integrate stress responses on reproduction. By isolating individual GnRH neurons from acute brain slices of adult female GnRH-EGFP transgenic mice and using microarray analyses, we have identified a range of transcripts encoding receptors known to be involved in stress responses in GnRH neurons. Prominent among these were receptors for corticotropin-releasing hormone (CRH), vasopressin, interleukins, prostaglandins, tumor necrosis factor alpha and other inflammatory mediators. We selected 4 of these targets [interleukin 1 receptor accessory protein (IL-1Racc), prostaglandin E(2) receptor subtype EP2 (PGER2), CRH receptor type 1 (CRH-R1), and arginine-vasopressin receptor type 1b (AVP-R1b)] for validation using single-cell RT-PCR from individual GnRH neurons. In total, 54% of GnRH neurons (n = 26) were found to express at least 1 of these transcripts. The IL-1Racc, PGER2 and CRH-R1 mRNAs were each detected in approximately 25% of the GnRH neurons tested, but no evidence was found for AVP-R1b transcripts. Overlap was found between the expression of CRH-R1 and PGER2, and IL-1Racc and PGER2 in individual GnRH neurons. Dual immunofluorescence experiments confirmed the expression of CRH-R1/2 in a subpopulation ( approximately 30%) of GnRH neurons. These observations indicate that a variety of different stressors and stress pathways have the capacity to have an impact directly upon a subpopulation of GnRH neurons to influence the reproductive axis.

  7. Analysis of Human mRNAs With the Reference Genome Sequence Reveals Potential Errors, Polymorphisms, and RNA Editing

    PubMed Central

    Furey, Terrence S.; Diekhans, Mark; Lu, Yontao; Graves, Tina A.; Oddy, Lachlan; Randall-Maher, Jennifer; Hillier, LaDeana W.; Wilson, Richard K.; Haussler, David

    2004-01-01

    The NCBI Reference Sequence (RefSeq) project and the NIH Mammalian Gene Collection (MGC) together define a set of ∼30,000 nonredundant human mRNA sequences with identified coding regions representing 17,000 distinct loci. These high-quality mRNA sequences allow for the identification of transcribed regions in the human genome sequence, and many researchers accept them as the correct representation of each defined gene sequence. Computational comparison of these mRNA sequences and the recently published essentially finished human genome sequence reveals several thousand undocumented nonsynonymous substitution and frame shift discrepancies between the two resources. Additional analysis is undertaken to verify that the euchromatic human genome is sufficiently complete—containing nearly the whole mRNA collection, thus allowing for a comprehensive analysis to be undertaken. Many of the discrepancies will prove to be genuine polymorphisms in the human population, somatic cell genomic variants, or examples of RNA editing. It is observed that the genome sequence variant has significant additional support from other mRNAs and ESTs, almost four times more often than does the mRNA variant, suggesting that the genome sequence is more accurate. In ∼15% of these cases, there is substantial support for both variants, suggestive of an undocumented polymorphism. An initial screening against a 24-individual genomic DNA diversity panel verified 60% of a small set of potential single nucleotide polymorphisms from which successful results could be obtained. We also find statistical evidence that a few of these discrepancies are due to RNA editing. Overall, these results suggest that the mRNA collections may contain a substantial number of errors. For current and future mRNA collections, it may be prudent to fully reconcile each genome sequence discrepancy, classifying each as a polymorphism, site of RNA editing or somatic cell variation, or genome sequence error. PMID:15489323

  8. Systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change.

    PubMed

    Xu, Qin; Song, Zhihong; Zhu, Caiyun; Tao, Chengcheng; Kang, Lifang; Liu, Wei; He, Fei; Yan, Juan; Sang, Tao

    2017-02-13

    Long non-coding RNA (lncRNA) is a class of non-coding RNA with important regulatory roles in biological process of organisms. The systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change are still poorly understood. Here we identified 17,610 lncRNAs and calculated their expression levels based on RNA-seq of 80 individuals of Miscanthus lutarioriparius from two environments, the nearly native habitats and transplanted field, respectively. LncRNAs had significantly higher expression diversity and lower expression frequency in population than protein coding mRNAs in both environments, which suggested that lncRNAs may experience more relaxed selection or divergent evolution in population compared with protein coding RNAs. In addition, the increase of expression diversity for lncRNAs was always significantly higher and the magnitude of fold change of expression in new stress environment was significantly larger than protein-coding mRNAs. These results suggested that lncRNAs may be more sensitive to environmental change than protein-coding mRNAs. Analysis of environment-robust and environment-specific lncRNA-mRNA co-expression network between two environments revealed the characterization of lncRNAs in response to environmental change. Furthermore, candidate lncRNAs contributing to water use efficiency (WUE) identified based on the WUE-lncRNA-mRNA co-expression network suggested the roles of lncRNAs in response to environmental change. Our study provided a comprehensive understanding of expression characterization of lncRNAs in population for M. lutarioriparius under field condition, which would be useful to explore the roles of lncRNAs and could accelerate the process of adaptation in new environment for many plants.

  9. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-05-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

  10. Protein disulfide isomerase as a regulator of chloroplast translational activation

    SciTech Connect

    Kim, Jungmook; Mayfield, S.P.

    1997-12-12

    Light-regulated translation of chloroplast messenger RNAs (mRNAs) requires transacting factors that interact with the 5{prime} untranslated region (UTR) of these mRNAs. Chloroplast polyadenylate-binding protein (cPABP) specifically binds to the 5{prime}-UTR of the psbA mRNA and is essential for translation of this mRNA. A protein disulfide isomeriase that is localized to the chloroplast and copurifies with cPABP was shown to modulate the binding of cPABP to the 5{prime}-UTR of the psbA mRNA by reversibly changing the redox status of cPaBP through redox potential or adenosine 5{prime}-diphosphate-dependent phosphorylation. This mechanism allows for a simple reversible switch regulating gene expression in the chloro