Science.gov

Sample records for puf9 regulates mrnas

  1. Regulation of maternal mRNAs in early development.

    PubMed

    Farley, Brian M; Ryder, Sean P

    2008-01-01

    Most sexually reproducing metazoans are anisogamous, meaning that the two gametes that combine during fertilization differ greatly in size. By convention, the larger gametes are considered female and are called ova, while the smaller gametes are male and are called sperm. In most cases, both gametes contribute similarly to the chromosomal content of the new organism. In contrast, the maternal gamete contributes nearly all of the cytoplasm. This cytoplasmic contribution is crucial to patterning early development; it contains the maternal proteins and transcripts that guide the early steps of development prior to the activation of zygotic transcription. This review compares and contrasts early development in common laboratory model organisms in order to highlight the similarities and differences in the regulation of maternal factors. We will focus on the production and reversible silencing of maternal mRNAs during oogenesis, their asymmetric activation after fertilization, and their subsequent clearance at the midblastula transition. Where possible, insights from mechanistic studies are presented. PMID:18365862

  2. Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs

    PubMed Central

    Willis, Dianna E.; van Niekerk, Erna A.; Sasaki, Yukio; Mesngon, Mariano; Merianda, Tanuja T.; Williams, Gervan G.; Kendall, Marvin; Smith, Deanna S.; Bassell, Gary J.; Twiss, Jeffery L.

    2007-01-01

    Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein–β-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous β-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs. PMID:17785519

  3. Regulation of Natural mRNAs by the Nonsense-Mediated mRNA Decay Pathway

    PubMed Central

    Peccarelli, Megan

    2014-01-01

    The nonsense-mediated mRNA decay (NMD) pathway is a specialized mRNA degradation pathway that degrades select mRNAs. This pathway is conserved in all eukaryotes examined so far, and it triggers the degradation of mRNAs that prematurely terminate translation. Originally identified as a pathway that degrades mRNAs with premature termination codons as a result of errors during transcription, splicing, or damage to the mRNA, NMD is now also recognized as a pathway that degrades some natural mRNAs. The degradation of natural mRNAs by NMD has been identified in multiple eukaryotes, including Saccharomyces cerevisiae, Drosophila melanogaster, Arabidopsis thaliana, and humans. S. cerevisiae is used extensively as a model to study natural mRNA regulation by NMD. Inactivation of the NMD pathway in S. cerevisiae affects approximately 10% of the transcriptome. Similar percentages of natural mRNAs in the D. melanogaster and human transcriptomes are also sensitive to the pathway, indicating that NMD is important for the regulation of gene expression in multiple organisms. NMD can either directly or indirectly regulate the decay rate of natural mRNAs. Direct NMD targets possess NMD-inducing features. This minireview focuses on the regulation of natural mRNAs by the NMD pathway, as well as the features demonstrated to target these mRNAs for decay by the pathway in S. cerevisiae. We also compare NMD-targeting features identified in S. cerevisiae with known NMD-targeting features in other eukaryotic organisms. PMID:25038084

  4. 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

  5. Subcellular Profiling Reveals Distinct and Developmentally Regulated Repertoire of Growth Cone mRNAs

    PubMed Central

    Zivraj, Krishna H.; Tung, Yi Chun Loraine; Piper, Michael; Gumy, Laura; Fawcett, James W.; Yeo, Giles S. H.; Holt, Christine E.

    2013-01-01

    Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons, yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Here laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genomewide microarray profiling. An unexpectedly large pool of mRNAs defined predominant pathways in protein synthesis, oxidative phosphorylation, cancer, neurological disease, and signaling. Comparative profiling of “young” (pathfinding) versus “old” (target-arriving) Xenopus growth cones revealed that the number and complexity of transcripts increases dramatically with age. Many presynaptic protein mRNAs are present exclusively in old growth cones, suggesting that functionally related sets of mRNAs are targeted to growth cones in a developmentally regulated way. Remarkably, a subset of mRNAs was significantly enriched in the growth cone compared with the axon compartment, indicating that mechanisms exist to localize mRNAs selectively to the growth cone. Furthermore, some receptor transcripts (e.g., EphB4), present exclusively in old growth cones, were equally abundant in young and old cell bodies, indicating that RNA trafficking from the soma is developmentally regulated. Our findings show that the mRNA repertoire in growth cones is regulated dynamically with age and suggest that mRNA localization is tailored to match the functional demands of the growing axon tip as it transforms into the presynaptic terminal. PMID:21084603

  6. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

    PubMed Central

    Poliseno, Laura; Salmena, Leonardo; Zhang, Jiangwen; Carver, Brett; Haveman, William J.; Pandolfi, Pier Paolo

    2011-01-01

    The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs possess a biological role in cancer cells that relies upon their ability to compete for microRNA binding and is independent of their protein-coding function. As a paradigm for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene (PTENP1) and the critical consequences of this interaction. We find that PTENP1 is biologically active as determined by its ability to regulate cellular levels of PTEN, and that it can exert a growth-suppressive role. We also show that PTENP1 locus is selectively lost in human cancer. We extend our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. Further, we demonstrate that the transcripts of protein coding genes such as PTEN are also biologically active. Together, these findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs. PMID:20577206

  7. Differential display of skin mRNAs regulated under varying environmental conditions in a mudskipper.

    PubMed

    Sakamoto, T; Yasunaga, H; Yokota, S; Ando, M

    2002-07-01

    To understand the molecular mechanisms underlying the terrestrial adaptation, as well as adaptation to different salinities, of the euryhaline and amphibious mudskipper ( Periophthalmus modestus), we have looked for the skin mRNAs that change during varying environmental conditions. Using differential mRNA display polymerase chain reaction, we compared skin mRNAs in mudskipper transferred from isotonic 30% seawater to fresh water or to seawater for 1 day and 7 days, as well as those kept out of water for 1 day. At the end of these periods, poly(A(+))RNA was prepared from the Cl(-)-secreting pectoral skins and also from the outer opercular skins where ion transport is negligible, and analyzed by differential display. We identified four cDNA products expressed differently under various environments as homologues of known genes. A further 34 cDNAs were expressed differentially, but they have no significant homology to identified sequences in GenBank. Northern blots demonstrate that mRNA levels of the actin-binding protein and the platelet-activating factor acetylhydrolase increased in the pectoral skins during seawater acclimation. The mRNA of the 90 kDa heat shock protein was down-regulated in water-deprived and freshwater fish, whose plasma cortisol levels were high. The aldolase mRNA was induced in both skins after desiccation. These four genes may be involved in the environmental adaptations. PMID:12122461

  8. Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs

    PubMed Central

    Tay, Yvonne; Kats, Lev; Salmena, Leonardo; Weiss, Dror; Tan, Shen Mynn; Ala, Ugo; Karreth, Florian; Poliseno, Laura; Provero, Paolo; Di Cunto, Ferdinando; Lieberman, Judy; Rigoutsos, Isidore; Pandolfi, Pier Paolo

    2011-01-01

    SUMMARY Here we demonstrate that protein-coding RNA transcripts can crosstalk by competing for common microRNAs, with microRNA response elements as the foundation of this interaction. We have termed such RNA transcripts as competing endogenous RNAs (ceRNAs). We tested this hypothesis in the context of PTEN, a key tumor suppressor whose abundance determines critical outcomes in tumorigenesis. By a combined computational and experimental approach, we identified and validated endogenous protein-coding transcripts that regulate PTEN, antagonize PI3K/AKT signaling and possess growth and tumor suppressive properties. Notably, we also show that these genes display concordant expression patterns with PTEN and copy number loss in cancers. Our study presents a road map for the prediction and validation of ceRNA activity and networks, and thus imparts a trans-regulatory function to protein-coding mRNAs. PMID:22000013

  9. IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs.

    PubMed

    Wang, Guangli; Huang, Zhenqiang; Liu, Xin; Huang, Wenhe; Chen, Shaoying; Zhou, Yanchun; Li, Deling; Singer, Robert H; Gu, Wei

    2016-03-29

    We have previously reported the ability of IMP1 in inhibiting proliferation and invasiveness of breast carcinoma cells in vitro. In the current study, we utilized a mouse xenograft model to further investigate the function of IMP1 in breast tumor progression and its underlying mechanism. We demonstrated that IMP1 expression significantly suppressed the growth of MDA231 cell-derived xenograft tumors and subsequent lung metastasis. Microarray analyses and differential gene expression identified handful mRNAs, many of which were involved in breast tumor-growth and metastasis. Further studies revealed that these mRNAs were directly interacted with the KH34 domain of IMP1 and this interaction post-transcriptionally regulated their corresponding protein expression. Either deletion of the KH34 domain of IMP1 or alteration of the expression of IMP1-bound mRNAs affected cell proliferation and tumor growth, producing the same phenotypes as IMP1 knockdown. Correlation of increased IMP1 expression with the reduced levels of its bound mRNAs, such as PTGS2, GDF15 and IGF-2 transcripts, was also observed in human breast tumors. Our studies provide insights into a molecular mechanism that the positive function of IMP1 to inhibit breast tumor growth and metastasis could be through the regulation of its target mRNAs. PMID:26910917

  10. IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

    PubMed Central

    Liu, Xin; Huang, Wenhe; Chen, Shaoying; Zhou, Yanchun; Li, Deling; Singer, Robert H.; Gu, Wei

    2016-01-01

    We have previously reported the ability of IMP1 in inhibiting proliferation and invasiveness of breast carcinoma cells in vitro. In the current study, we utilized a mouse xenograft model to further investigate the function of IMP1 in breast tumor progression and its underlying mechanism. We demonstrated that IMP1 expression significantly suppressed the growth of MDA231 cell-derived xenograft tumors and subsequent lung metastasis. Microarray analyses and differential gene expression identified handful mRNAs, many of which were involved in breast tumor-growth and metastasis. Further studies revealed that these mRNAs were directly interacted with the KH34 domain of IMP1 and this interaction post-transcriptionally regulated their corresponding protein expression. Either deletion of the KH34 domain of IMP1 or alteration of the expression of IMP1-bound mRNAs affected cell proliferation and tumor growth, producing the same phenotypes as IMP1 knockdown. Correlation of increased IMP1 expression with the reduced levels of its bound mRNAs, such as PTGS2, GDF15 and IGF-2 transcripts, was also observed in human breast tumors. Our studies provide insights into a molecular mechanism that the positive function of IMP1 to inhibit breast tumor growth and metastasis could be through the regulation of its target mRNAs. PMID:26910917

  11. 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

  12. Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression.

    PubMed

    Miniard, Angela C; Middleton, Lisa M; Budiman, Michael E; Gerber, Carri A; Driscoll, Donna M

    2010-08-01

    Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3' untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level. PMID:20385601

  13. The PARN Deadenylase Targets a Discrete Set of mRNAs for Decay and Regulates Cell Motility in Mouse Myoblasts

    PubMed Central

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

    2012-01-01

    PARN is one of several deadenylase enzymes present in mammalian cells, and as such the contribution it makes to the regulation of gene expression is unclear. To address this, we performed global mRNA expression and half-life analysis on mouse myoblasts depleted of PARN. PARN knockdown resulted in the stabilization of 40 mRNAs, including that encoding the mRNA decay factor ZFP36L2. Additional experiments demonstrated that PARN knockdown induced an increase in Zfp36l2 poly(A) tail length as well as increased translation. The elements responsible for PARN-dependent regulation lie within the 3′ UTR of the mRNA. Surprisingly, changes in mRNA stability showed an inverse correlation with mRNA abundance; stabilized transcripts showed either no change or a decrease in mRNA abundance. Moreover, we found that stabilized mRNAs had reduced accumulation of pre–mRNA, consistent with lower transcription rates. This presents compelling evidence for the coupling of mRNA decay and transcription to buffer mRNA abundances. Although PARN knockdown altered decay of relatively few mRNAs, there was a much larger effect on global gene expression. Many of the mRNAs whose abundance was reduced by PARN knockdown encode factors required for cell migration and adhesion. The biological relevance of this observation was demonstrated by the fact that PARN KD cells migrate faster in wound-healing assays. Collectively, these data indicate that PARN modulates decay of a defined set of mRNAs in mammalian cells and implicate this deadenylase in coordinating control of genes required for cell movement. PMID:22956911

  14. CRM 1-Mediated Degradation and Agonist-Induced Down-Regulation of β-Adrenergic Receptor mRNAs

    PubMed Central

    Bai, Ying; Lu, Huafei; Machida, Curtis A.

    2006-01-01

    SUMMARY The β1-adrenergic receptor (β1-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 β1-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 β1-ARs in the brain, results in significant decreases in β1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in β1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the β1-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 β1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the β1-AR mRNAs. PMID:16997396

  15. Divergent protein coding regions in otherwise closely related androgen-regulated mRNAs.

    PubMed Central

    McDonald, C J; Eliopoulos, E; Higgins, S J

    1984-01-01

    Rat seminal vesicles serve as a model system for studying androgen action. We have sequenced and compared full length cDNAs for two major proteins (S and F) synthesised and secreted under hormonal control. Overall, mRNAS and mRNAF share 57% nucleotide sequence homology suggesting that their genes arose by duplication of a common ancestor. However, the mRNAs display a highly unusual regional distribution of sequence homology, with the untranslated regions (UTRs) being substantially more homologous than the protein-coding regions (PCRs). Detailed analysis of nucleotide substitutions at synonymous and replacement sites shows that the PCRs have evolved very rapidly. Evolutionary conservation of the UTRs is no higher than that of UTRs generally and thus provides no evidence of a specific regulatory role for the UTRs in androgen action. The primary sequences of proteins S and F have diverged so rapidly that they are the best examples of neutrally evolving proteins for which comparative nucleotide sequence data are available. However, despite their rapid divergence, the predicted higher order structures for both proteins consist largely of non-regular conformation. This is discussed in terms of their roles as structural components of the rodent copulatory plug. PMID:6548962

  16. 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. PMID:26283043

  17. 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.

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

    PubMed Central

    Khan, Israr; Kerwin, John; Owen, Kate; Griner, Erin; Iorns, Elizabeth

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.08245.001 PMID:26335297

  19. 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.

  20. 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

  1. Regulation of smooth muscle contractility by competing endogenous mRNAs in intracranial aneurysms.

    PubMed

    Zhang, Mingming; Ren, Yuan; Wang, Yajie; Wang, Renzhi; Zhou, Qian; Peng, Yong; Li, Qi; Yu, Mengqiang; Jiang, Yugang

    2015-05-01

    Alterations in vascular smooth muscle cells (SMCs) contribute to the pathogenesis of intracranial aneurysms (IAs), but the genetic mechanisms underlying these alterations are unclear. We used microarray analysis to compare tissue small noncoding RNA and messenger RNA expression profiles in vessel wall samples from patients with late-stage IAs. We identified myocardin (MYOCD), a key contractility regulator of vascular SMCs, as a critical factor in IA progression. Using a multifaceted computational and experimental approach, we determined that depletion of competitive endogenous RNAs (ARHGEF12, FGF12, and ADCY5) enhanced factors that downregulate MYOCD, which induces the conversion of SMCs from differentiated contractile states into dedifferentiated phenotypes that exhibit enhanced proliferation, synthesis of new extracellular matrix, and organization of mural thrombi. These effects may lead to the repair and maintenance of IAs. This study presents guidelines for the prediction and validation of the IA regulator MYOCD in competitive endogenous RNA networks and facilitates the development of novel therapeutic and diagnostic tools for IAs. PMID:25868147

  2. Zonisamide up-regulated the mRNAs encoding astrocytic anti-oxidative and neurotrophic factors.

    PubMed

    Choudhury, M E; Sugimoto, K; Kubo, M; Iwaki, H; Tsujii, T; Kyaw, W T; Nishikawa, N; Nagai, M; Tanaka, J; Nomoto, M

    2012-08-15

    Zonisamide has been proven as an effective drug for the recovery of degenerating dopaminergic neurons in the animal models of Parkinson's disease. However, several lines of evidence have questioned the neuroprotective capacity of zonisamide in animal models of Parkinson's disease. Although it suppresses dopaminergic neurodegeneration in animal models, the cellular and molecular mechanisms underlying the effectiveness of zonisamide are not fully understood. The current study demonstrates the effects of zonisamide on astrocyte cultures and two 6-hydroxydopamine-induced models of Parkinson's disease. Using primary astrocyte cultures, we showed that zonisamide up-regulated the expression of mRNA encoding mesencephalic astrocyte-derived neurotrophic factor, vascular endothelial growth factor, proliferating cell nuclear antigen, metallothionein-2, copper/zinc superoxide dismutase, and manganese superoxide dismutase. Similar responses to zonisamide were found in substantia nigra where the rats were pre-treated with 6-hydroxydopamine. Notably, pharmacological inhibition of 6-hydroxydopamine-induced toxicity by zonisamide pre-treatment was also confirmed using rat mesencephalic organotypic slice cultures of substantia nigra. In addition to this, zonisamide post-treatment also attenuated the nigral tyrosine hydroxylase-positive neuronal loss induced by 6-hydroxydopamine. Taken together, these studies demonstrate that zonisamide protected dopamine neurons in two Parkinson's disease models through a novel mechanism, namely increasing the expression of some important astrocyte-mediated neurotrophic and anti-oxidative factors. PMID:22659113

  3. Registered report: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs

    PubMed Central

    Phelps, Mitch; Coss, Chris; Wang, Hongyan; Cook, Matthew

    2016-01-01

    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 “Coding-Independent Regulation of the Tumor Suppressor PTEN by Competing Endogenous 'mRNAs' by Tay and colleagues, published in Cell in 2011 (Tay et al., 2011). The experiments to be replicated are those reported in Figures 3C, 3D, 3G, 3H, 5A and 5B, and in Supplemental Figures 3A and B. Tay and colleagues proposed a new regulatory mechanism based on competing endogenous RNAs (ceRNAs), which regulate target genes by competitive binding of shared microRNAs. They test their model by identifying and confirming ceRNAs that target PTEN. In Figure 3A and B, they report that perturbing expression of putative PTEN ceRNAs affects expression of PTEN. This effect is dependent on functional microRNA machinery (Figure 3G and H), and affects the pathway downstream of PTEN itself (Figures 5A and B). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.12470.001 PMID:26943900

  4. A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites

    PubMed Central

    Sharma, Cynthia M.; Darfeuille, Fabien; Plantinga, Titia H.; Vogel, Jörg

    2007-01-01

    The interactions of numerous regulatory small RNAs (sRNAs) with target mRNAs have been characterized, but how sRNAs can regulate multiple, structurally unrelated mRNAs is less understood. Here we show that Salmonella GcvB sRNA directly acts on seven target mRNAs that commonly encode periplasmic substrate-binding proteins of ABC uptake systems for amino acids and peptides. Alignment of GcvB homologs of distantly related bacteria revealed a conserved G/U-rich element that is strictly required for GcvB target recognition. Analysis of target gene fusion regulation in vivo, and in vitro structure probing and translation assays showed that GcvB represses its target mRNAs by binding to extended C/A-rich regions, which may also serve as translational enhancer elements. In some cases (oppA, dppA), GcvB repression can be explained by masking the ribosome-binding site (RBS) to prevent 30S subunit binding. However, GcvB can also effectively repress translation by binding to target mRNAs at upstream sites, outside the RBS. Specifically, GcvB represses gltI mRNA translation at the C/A-rich target site located at positions −57 to −45 relative to the start codon. Taken together, our study suggests highly conserved regions in sRNAs and mRNA regions distant from Shine-Dalgarno sequences as important elements for the identification of sRNA targets. PMID:17974919

  5. 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

  6. Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation

    PubMed Central

    Graf, Robin; Munschauer, Mathias; Mastrobuoni, Guido; Mayr, Florian; Heinemann, Udo; Kempa, Stefan; Rajewsky, Nikolaus; Landthaler, Markus

    2013-01-01

    The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA-binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B-binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28-binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knockdown, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts. PMID:23770886

  7. CstF64: Cell Cycle Regulation and Functional Role in 3′ End Processing of Replication-Dependent Histone mRNAs

    PubMed Central

    Romeo, Valentina; Griesbach, Esther

    2014-01-01

    The 3′ end processing of animal replication-dependent histone mRNAs is activated during G1/S-phase transition. The processing site is recognized by stem-loop binding protein and the U7 snRNP, but cleavage additionally requires a heat-labile factor (HLF), composed of cleavage/polyadenylation specificity factor, symplekin, and cleavage stimulation factor 64 (CstF64). Although HLF has been shown to be cell cycle regulated, the mechanism of this regulation is unknown. Here we show that levels of CstF64 increase toward the S phase and its depletion affects histone RNA processing, S-phase progression, and cell proliferation. Moreover, analyses of the interactions between CstF64, symplekin, and the U7 snRNP-associated proteins FLASH and Lsm11 indicate that CstF64 is important for recruiting HLF to histone precursor mRNA (pre-mRNA)-resident proteins. Thus, CstF64 is central to the function of HLF and appears to be at least partly responsible for its cell cycle regulation. Additionally, we show that misprocessed histone transcripts generated upon CstF64 depletion mainly accumulate in the nucleus, where they are targets of the exosome machinery, while a small cytoplasmic fraction is partly associated with polysomes. PMID:25266659

  8. 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

  9. Stabilization of cellular mRNAs and up-regulation of proteins by oligoribonucleotides homologous to the Bcl2 adenine-uridine rich element motif.

    PubMed

    Bevilacqua, Annamaria; Ghisolfi, Laura; Franzi, Sara; Maresca, Giovanna; Gherzi, Roberto; Capaccioli, Sergio; Nicolin, Angelo; Canti, Gianfranco

    2007-02-01

    Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs. PMID:17077270

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

    PubMed

    Nicolas, Francisco Esteban; Moxon, Simon; de Haro, Juan P; Calo, Silvia; Grigoriev, Igor V; Torres-Martínez, Santiago; Moulton, Vincent; Ruiz-Vázquez, Rosa M; Dalmay, Tamas

    2010-09-01

    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. PMID:20427422

  11. 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

  12. 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

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

    NASA Astrophysics Data System (ADS)

    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.

  14. 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

  15. Coordinate regulation of a family of promastigote-enriched mRNAs by the 3′UTR PRE element in Leishmania mexicana

    PubMed Central

    Holzer, Timothy R.; Mishra, Krishna K.; LeBowitz, Jonathan H.; Forney, James D.

    2009-01-01

    Post-transcriptional regulation is a key feature controlling gene expression in the protozoan parasite Leishmania. The nine-nucleotide paraflagellar rod regulatory element (PRE) in the 3′UTR of L. mexicana PFR2 is both necessary and sufficient for the observed ten-fold higher level of PFR2 mRNA in promastigotes compared to amastigotes. It is also found in the 3′UTRs of all known PFR genes. A search of the L. major Friedlin genomic database revealed several genes that share this cis element including a homolog of a heterotrimeric kinesin II subunit, and a gene that shares identity to a homolog of a Plasmodium antigen. In this study, we show that genes that harbor the PRE display promastigote-enriched transcript accumulation ranging from 4 – 15 fold. Northern analysis on episomal block substitution constructs revealed that the regulatory element is necessary for the proper steady-state accumulation of mRNA in L. mexicana paraflagellar rod gene 4 (PFR4). Also we show that the PRE plays a major role in the proper steady-state mRNA accumulation of PFR1, but may not account for the full regulatory mechanism acting on this mRNA. Our evidence suggests that the PRE coordinately regulates the mRNA abundance of not only the PFR family of genes, but in a larger group of genes that have unrelated functions. Although the PRE alone can regulate some mRNAs, it may also act in concert with additional elements to control other RNA transcripts. PMID:18023890

  16. 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.

  17. 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. PMID:27606618

  18. Untranslated regions of mRNAs

    PubMed Central

    Mignone, Flavio; Gissi, Carmela; Liuni, Sabino; Pesole, Graziano

    2002-01-01

    Gene expression is finely regulated at the post-transcriptional level. Features of the untranslated regions of mRNAs that control their translation, degradation and localization include stem-loop structures, upstream initiation codons and open reading frames, internal ribosome entry sites and various cis-acting elements that are bound by RNA-binding proteins. PMID:11897027

  19. 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. PMID:23382661

  20. 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

  1. Genetic analysis of the phenobarbital regulation of the cytochrome P-450 2b-9 and aldehyde dehydrogenase type 2 mRNAs in mouse liver.

    PubMed Central

    Damon, M; Fautrel, A; Guillouzo, A; Corcos, L

    1996-01-01

    The aim of this study was to investigate the effect of the genetic background on the phenobarbital inducibility of cytochrome P-450 2b-9, cytochrome P-450 2b-10 and aldehyde dehydrogenase type 2 mRNAs in mice. We analysed the basal expression and the phenobarbital inducibility of both cytochrome P-450 mRNAs by semi-quantitative specific reverse transcription-PCR analyses in five inbred mouse strains (A/J,BALB/cByJ,C57BL/6J, DBA/2J and SWR/J). Male mice constitutively expressed cytochrome P-450 2b-9 and cytochrome P-450 2b-10 mRNAs, but a number of differences in their response to phenobarbital were observed. In all these mouse strains, phenobarbital induced cytochrome P-450 2b-10 mRNA whereas it could have either a positive or a negative effect on cytochrome P-450 2b-9 expression, depending on the strain and the sex of the mice. Specifically, phenobarbital increased cytochrome P-450 2b-9 expression in C57BL/6J males while it decreased it in DBA/2J mice. Interestingly, dexamethasone was able to mimic the phenobarbital effect on both cytochromes P-450 in these two strains. Aldehyde dehydrogenase type 2 mRNA was always induced by phenobarbital, except in the C57BL/6J strain. Genetic analysis revealed that the phenobarbital-inducible phenotype was either a semi-dominant or a recessive trait in F1 animals from a C57BL/6J x DBA/2J cross for the cytochrome P-450 2b-9 and the aldehyde dehydrogenase type 2 genes, respectively. This study suggests that the genetic basis for phenobarbital induction in mice depends on the target gene, and that more than one regulatory step would by involved in this response pathway. PMID:8713075

  2. Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats.

    PubMed

    Laping, N J; Nichols, N R; Day, J R; Finch, C E

    1991-07-01

    This study examined the effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the levels of two astrocyte mRNAs during responses to unilateral entorhinal cortex lesions (ECL) to identify molecular mechanisms involved in glucocorticoid modulation of astrocyte activation following deafferentation. Both glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) mRNA were increased in the ipsilateral hippocampus 4 days following unilateral ECL. In unlesioned ADX rats CORT replacement decreased both messages in the hippocampus. CORT replacement suppressed the ECL-induced increase of GFAP mRNA in the contralateral, but not ipsilateral hippocampus of ADX rats. In contrast, CORT decreased SGP-2 mRNA both ipsi- and contralaterally. It is clear that several regulatory mechanisms are responsible for maintaining a physiological balance of astrocyte activity in the adult brain, and that changes in circuit integrity and the endocrine milieu can alter this balance. PMID:1717807

  3. Genome-wide analysis reveals the differential regulations of mRNAs and miRNAs in Dorset and Small Tail Han sheep muscles.

    PubMed

    Miao, Xiangyang; Luo, Qingmiao; Qin, Xiaoyu

    2015-05-15

    Sheep are highly diverse species raised for meat and other agricultural products. The aim of the present study was to investigate the genetic regulators that could control muscle growth and development in different sheep breeds. The study showed that the differentially expressed genes are involved in various cellular activities, such as metabolic cascades, catalytic function and signaling pathway. Many signaling molecules are also found to be differentially expressed, suggesting important roles of signaling pathways contributing to genetic diversity and sheep development. Analysis of miRNAs suggested important roles of miRNAs in controlling muscle differences. This study provided a genome-wide resolution of mRNA and miRNA regulations in muscles from Dorset and Han sheep. PMID:25732516

  4. Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism.

    PubMed Central

    Godt, D E; Roitsch, T

    1997-01-01

    The aim of the present study was to gain insight into the contribution of extracellular invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ. PMID:9306701

  5. 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

    2016-01-01

    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. PMID:27173348

  6. mRNAs Hit a Sticky Wicket.

    PubMed

    Voronina, Ekaterina

    2016-04-01

    Drosophila germ cell specification depends on localization of mRNAs required for patterning to the posterior of the oocyte during oogenesis. In a recent issue of Nature, Vourekas et al. (2016) suggest that Aubergine in complex with piRNAs may provide a low-specificity anchoring mechanism for posterior mRNAs. PMID:27046827

  7. Differential distribution of ELMO1 and ELMO2 mRNAs in the developing mouse brain.

    PubMed

    Katoh, Hironori; Fujimoto, Satoshi; Ishida, Chisaki; Ishikawa, Yukio; Negishi, Manabu

    2006-02-16

    ELMO is an upstream regulator of the Rho family small GTPase Rac. We investigated the distributions of mRNAs of two subtypes of ELMO, ELMO1 and ELMO2, in the developing mouse brain. Both ELMO1 and ELMO2 mRNAs are widely distributed in the developing mouse brain, but they were expressed in different neuronal populations in the cerebral cortex, thalamus, and cerebellum. Thus, ELMO1 and ELMO2 may play different roles during brain development. PMID:16443196

  8. Ribosome association contributes to restricting mRNAs to the cell body of hippocampal neurons.

    PubMed

    Lu, Z; McLaren, R S; Winters, C A; Ralston, E

    1998-12-01

    In neurons, mRNAs are differentially sorted to axons, dendrites, and the cell body. Recently, regions of certain mRNAs have been identified that target those mRNAs for translocation to the processes. However, the mechanism by which many, if not most mRNAs are retained in the cell body is not understood. Total inhibition of translation, by puromycin or cycloheximide, results in the mislocalization of cell body mRNAs to dendrites. We have examined the effect of translational inhibitors on the localization of ferritin mRNA, the translation of which can also be inhibited specifically by reducing iron levels. Using nonisotopic in situ hybridization, ferritin mRNA is found restricted to the cell body of cultured rat hippocampal neurons. Following treatment with either puromycin or cycloheximide, it migrates into dendrites. Control experiments reveal that the drugs affect neither the viability of the neuronal cultures, nor the steady-state level of ferritin mRNA. When transcription and protein synthesis are inhibited simultaneously, ferritin mRNA is found in the dendrites of puromycin, but not of cycloheximide-treated neurons. However, the localization of ferritin mRNA is unaffected by changes in iron concentration that regulate its translation rate specifically. We propose a model whereby cell body-restricted mRNAs are maintained in that location by association with ribosomes and with another cell component, which traps mRNAs when they are freed of ribosome association. The release of all mRNA species, as happens after total protein synthesis inhibition, floods the system and allows cell body mRNAs to diffuse into dendrites. In contrast, the partial release of the single ferritin mRNA species does not saturate the trapping system and the mRNA is retained in the cell body. PMID:9888989

  9. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

    PubMed Central

    Elmén, Joacim; Lindow, Morten; Silahtaroglu, Asli; Bak, Mads; Christensen, Mette; Lind-Thomsen, Allan; Hedtjärn, Maj; Hansen, Jens Bo; Hansen, Henrik Frydenlund; Straarup, Ellen Marie; McCullagh, Keith; Kearney, Phil; Kauppinen, Sakari

    2008-01-01

    MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context. PMID:18158304

  10. MicroRNA-mediated repression of nonsense mRNAs

    PubMed Central

    Zhao, Ya; Lin, Jimin; Xu, Beiying; Hu, Sida; Zhang, Xue; Wu, Ligang

    2014-01-01

    Numerous studies have established important roles for microRNAs (miRNAs) in regulating gene expression. Here, we report that miRNAs also serve as a surveillance system to repress the expression of nonsense mRNAs that may produce harmful truncated proteins. Upon recognition of the premature termination codon by the translating ribosome, the downstream portion of the coding region of an mRNA is redefined as part of the 3′ untranslated region; as a result, the miRNA-responsive elements embedded in this region can be detected by miRNAs, triggering accelerated mRNA deadenylation and translational inhibition. We demonstrate that naturally occurring cancer-causing APC (adenomatous polyposis coli) nonsense mutants which escape nonsense-mediated mRNA decay (NMD) are repressed by miRNA-mediated surveillance. In addition, we show that miRNA-mediated surveillance and exon–exon junction complex-mediated NMD are not mutually exclusive and act additively to enhance the repressive activity. Therefore, we have uncovered a new role for miRNAs in repressing nonsense mutant mRNAs. DOI: http://dx.doi.org/10.7554/eLife.03032.001 PMID:25107276

  11. 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

  12. Proteomic analysis reveals the dynamic association of proteins with translated mRNAs in Trypanosoma cruzi.

    PubMed

    Alves, Lysangela R; Avila, Andréa R; Correa, Alejandro; Holetz, Fabíola B; Mansur, Fernanda C B; Manque, Patrício A; de Menezes, Juliana P B; Buck, Gregory A; Krieger, Marco A; Goldenberg, Samuel

    2010-03-01

    Gene regulation is mainly post-transcriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Post-transcriptional regulation requires the association between mRNAs and certain proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poly(T) beads to isolate and characterize proteins and protein complexes bound to poly-A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC-MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and post-polysomal (2.95%) fractions; stress polysomal (13.8%) and post-polysomal (40.78%) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi. PMID:20060445

  13. Hormonal regulation of rat hypothalamic neuropeptide mRNAs: effect of hypophysectomy and hormone replacement on growth-hormone-releasing factor, somatostatin and the insulin-like growth factors.

    PubMed

    Wood, T L; Berelowitz, M; Gelato, M C; Roberts, C T; LeRoith, D; Millard, W J; McKelvy, J F

    1991-03-01

    Hormonal feedback regulation of hypothalamic peptides putatively involved in growth hormone (GH) regulation has been studied by measurement of steady-state mRNA levels in male hypophysectomized rats with or without thyroid hormone, corticosterone, testosterone or GH replacement. Hypothalamic GH-releasing factor (GRF) mRNA levels increased progressively following hypophysectomy to 420% of sham levels after 15 days while hypothalamic insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) mRNA levels decreased to less than 40% of sham levels. Whole hypothalamic somatostatin mRNA levels were not significantly different from sham. One week of continuous GH infusion restored hypothalamic IGF-I mRNA to levels (95%) indistinguishable from those in sham-operated controls but had no effect on either IGF-II or GRF mRNA. Thyroid hormone, corticosterone and testosterone treatment without GH had no effect on the hypophysectomy-induced reduction of either IGF-I or IGF-II mRNA levels but reversed the elevation of GRF mRNA. We conclude that hypothalamic IGF-I may be involved in GH feedback regulation and thus may function as a hypothalamic modulator of GH. In contrast, IGF-II may be regulated by one of the pituitary trophic hormones but not by GH or the target hormones tested. Finally, hypothalamic GRF mRNA regulation appears to be complex and may include target hormone feedback. PMID:1674982

  14. 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

  15. Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo

    PubMed Central

    Barckmann, Bridlin; Pierson, Stéphanie; Dufourt, Jérémy; Papin, Catherine; Armenise, Claudia; Port, Fillip; Grentzinger, Thomas; Chambeyron, Séverine; Baronian, Grégory; Desvignes, Jean-Pierre; Curk, Tomaz; Simonelig, Martine

    2015-01-01

    Summary The Piwi-interacting RNA (piRNA) pathway plays an essential role in the repression of transposons in the germline. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. Here, we perform iCLIP with the PIWI protein Aubergine (Aub) and identify hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants. PMID:26257181

  16. Nuleclear extracts of Crithidia fasciculata contain a factor(s) that binds to the 5'-untranslated regions of TOP2 and RPA1 mRNAs containing sequences required for their cell cycle regulation.

    PubMed

    Mahmood, R; Ray, D S

    1998-09-11

    The Crithidia fasciculata replication protein A gene, RPA1, and topoisomerase II gene, TOP2, encode proteins involved in the replication of nuclear and mitochondrial DNA, respectively. Transcripts of both genes accumulate periodically during the cell cycle and attain their maximum levels just before S phase. Octamer consensus sequences within the 5'-untranslated region (UTR) of both genes have been shown to be necessary for cycling of these transcripts. Using a gel retardation assay, we show here that nuclear extracts of C. fasciculata contain a protein factor(s) that binds specifically to RNA from 5'-UTRs of TOP2 and RPA1 genes. In addition, mutations in the consensus octamer sequence abolish binding to the RNA in both cases. Ultraviolet cross-linking using a radiolabeled TOP2 5'-UTR probe identified proteins with apparent molecular masses of 74 and 37 kDa in the RNA-protein complex. Nuclear extracts prepared from synchronized cells show that the binding activity varies during the cell cycle in parallel with TOP2 and RPA1 mRNA levels. These results suggest that the cell cycle regulation of the mRNA levels of trypanosomatid DNA replication genes may be mediated by binding of specific proteins to conserved sequences in the 5'-UTR of their transcripts. PMID:9726980

  17. Dietary sucrose enhances processing of mRNA-S14 nuclear precursor.

    PubMed

    Burmeister, L A; Mariash, C N

    1991-12-01

    The rapid response of rat hepatic mRNA-S14 to hormonal or dietary manipulation makes it an excellent model to study the control of lipogenic enzyme mRNA. The mechanism of regulation of this mRNA by triiodothyronine (T3) or sucrose remains controversial. Although initial studies suggested that T3 stabilized the nuclear precursor, subsequent studies suggest that T3 acts by increasing the transcriptional rate of this gene. More recently, the induction of mRNA-S14 by sucrose administration was shown to be associated with an increase in transcriptional "run-on" activity. Because T3 and carbohydrate feeding synergistically regulate this mRNA, we studied the response to short and long term high carbohydrate feeding in hypothyroid and euthyroid rats. We found the response to the lipogenic diet was rapid in hypothyroid rats, with maximal levels of mRNA-S14 attained by 4 h (2.2 +/- 0.6 chow fed versus 13.5 +/- 2.5 pg/micrograms RNA on lipogenic diet). The rapid induction by the lipogenic diet contrasts with the diminished response to sucrose by gastric gavage (4.6 +/- 1.2 pg/micrograms RNA) over the same time interval. Despite the large increase in the mature mRNA induced by the lipogenic diet, the rise in the nuclear precursor was small and not different from that observed after sucrose gavage (0.14 +/- 0.01 chow, 0.26 +/- 0.03 sucrose gavage, 0.25 +/- 0.04 pg/micrograms RNA lipogenic diet). The molar ratio of the mature to precursor mRNA-S14 showed progressive increases with the smallest level in the fasting rat, an intermediate level in the chow-fed and sucrose gavaged rats, and the highest level in the animals fed a lipogenic diet (2.1, 16.5, 16.3, 62.7, respectively). Based on the previously reported half-life for the mature mRNA-S14, these data show that feeding sucrose by gavage or by a lipogenic diet leads to enhanced fractional conversion of precursor to mature mRNA-S14 with a simultaneous stabilization of the precursor mRNA-S14. PMID:1744084

  18. Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

    PubMed Central

    Lavut, Anna; Raveh, Dina

    2012-01-01

    Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance

  19. A beacon in the cytoplasm: Tracking translation of single mRNAs.

    PubMed

    Pingali, Hema V; Hilliker, Angela K

    2016-09-12

    Translation is carefully regulated to control protein levels and allow quick responses to changes in the environment. Certain questions about translation in vivo have been unattainable until now. In this issue, Pichon et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201605024) describe a new technique to allow real-time monitoring of translation on single mRNAs. PMID:27597752

  20. Protein arginine methyltransferase CARM1 attenuates the paraspeckle-mediated nuclear retention of mRNAs containing IRAlus.

    PubMed

    Hu, Shi-Bin; Xiang, Jian-Feng; Li, Xiang; Xu, Yefen; Xue, Wei; Huang, Min; Wong, Catharine C; Sagum, Cari A; Bedford, Mark T; Yang, Li; Cheng, Donghang; Chen, Ling-Ling

    2015-03-15

    In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3' untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54(nrb). However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54(nrb), resulting in reduced binding of p54(nrb) to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein-RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1. PMID:25792598

  1. Protein arginine methyltransferase CARM1 attenuates the paraspeckle-mediated nuclear retention of mRNAs containing IRAlus

    PubMed Central

    Hu, Shi-Bin; Xiang, Jian-Feng; Li, Xiang; Xu, Yefen; Xue, Wei; Huang, Min; Wong, Catharine C.; Sagum, Cari A.; Bedford, Mark T.; Yang, Li

    2015-01-01

    In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3′ untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54nrb. However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54nrb, resulting in reduced binding of p54nrb to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein–RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1. PMID:25792598

  2. Alternative Mechanisms to Initiate Translation in Eukaryotic mRNAs

    PubMed Central

    Martínez-Salas, Encarnación; Piñeiro, David; Fernández, Noemí

    2012-01-01

    The composition of the cellular proteome is under the control of multiple processes, one of the most important being translation initiation. The majority of eukaryotic cellular mRNAs initiates translation by the cap-dependent or scanning mode of translation initiation, a mechanism that depends on the recognition of the m7G(5′)ppp(5′)N, known as the cap. However, mRNAs encoding proteins required for cell survival under stress bypass conditions inhibitory to cap-dependent translation; these mRNAs often harbor internal ribosome entry site (IRES) elements in their 5′UTRs that mediate internal initiation of translation. This mechanism is also exploited by mRNAs expressed from the genome of viruses infecting eukaryotic cells. In this paper we discuss recent advances in understanding alternative ways to initiate translation across eukaryotic organisms. PMID:22536116

  3. 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. PMID:26984228

  4. 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

  5. Uridylation prevents 3′ trimming of oligoadenylated mRNAs

    PubMed Central

    Sement, François Michaël; Ferrier, Emilie; Zuber, Hélène; Merret, Rémy; Alioua, Malek; Deragon, Jean-Marc; Bousquet-Antonelli, Cécile; Lange, Heike; Gagliardi, Dominique

    2013-01-01

    Degradation of mRNAs is usually initiated by deadenylation, the shortening of long poly(A) tails to oligo(A) tails of 12–15 As. Deadenylation leads to decapping and to subsequent 5′ to 3′ degradation by XRN proteins, or alternatively 3′ to 5′ degradation by the exosome. Decapping can also be induced by uridylation as shown for the non-polyadenylated histone mRNAs in humans and for several mRNAs in Schizosaccharomyces pombe and Aspergillus nidulans. Here we report a novel role for uridylation in preventing 3′ trimming of oligoadenylated mRNAs in Arabidopsis. We show that oligo(A)-tailed mRNAs are uridylated by the cytosolic UTP:RNA uridylyltransferase URT1 and that URT1 has no major impact on mRNA degradation rates. However, in absence of uridylation, oligo(A) tails are trimmed, indicating that uridylation protects oligoadenylated mRNAs from 3′ ribonucleolytic attacks. This conclusion is further supported by an increase in 3′ truncated transcripts detected in urt1 mutants. We propose that preventing 3′ trimming of oligo(A)-tailed mRNAs by uridylation participates in establishing the 5′ to 3′ directionality of mRNA degradation. Importantly, uridylation prevents 3′ shortening of mRNAs associated with polysomes, suggesting that a key biological function of uridylation is to confer 5′ to 3′ polarity in case of co-translational mRNA decay. PMID:23748567

  6. 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. PMID:25941326

  7. 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

  8. 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

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

    PubMed Central

    2014-01-01

    Background 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. Results 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. Conclusions 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. PMID:24898526

  10. Exon Junction Complexes Show a Distributional Bias toward Alternatively Spliced mRNAs and against mRNAs Coding for Ribosomal Proteins.

    PubMed

    Hauer, Christian; Sieber, Jana; Schwarzl, Thomas; Hollerer, Ina; Curk, Tomaz; Alleaume, Anne-Marie; Hentze, Matthias W; Kulozik, Andreas E

    2016-08-01

    The exon junction complex (EJC) connects spliced mRNAs to posttranscriptional processes including RNA localization, transport, and regulated degradation. Here, we provide a comprehensive analysis of bona fide EJC binding sites across the transcriptome including all four RNA binding EJC components eIF4A3, BTZ, UPF3B, and RNPS1. Integration of these data sets permits definition of high-confidence EJC deposition sites as well as assessment of whether EJC heterogeneity drives alternative nonsense-mediated mRNA decay pathways. Notably, BTZ (MLN51 or CASC3) emerges as the EJC subunit that is almost exclusively bound to sites 20-24 nucleotides upstream of exon-exon junctions, hence defining EJC positions. By contrast, eIF4A3, UPF3B, and RNPS1 display additional RNA binding sites suggesting accompanying non-EJC functions. Finally, our data show that EJCs are largely distributed across spliced RNAs in an orthodox fashion, with two notable exceptions: an EJC deposition bias in favor of alternatively spliced transcripts and against the mRNAs that encode ribosomal proteins. PMID:27475226

  11. mRNAs from human adenovirus 2 early region 4.

    PubMed Central

    Virtanen, A; Gilardi, P; Näslund, A; LeMoullec, J M; Pettersson, U; Perricaudet, M

    1984-01-01

    The molecular structure of the mRNAs from early region 4 of human adenovirus 2 has been studied by Northern blot analysis, S1 nuclease analysis, and sequence analysis of cDNA clones. The results make it possible to identify four different splice donor sites and six different splice acceptor sites. The structure of 12 different mRNAs can be deduced from the analysis. The mRNAs have identical 5' and 3' ends and are thus likely to be processed from a common mRNA precursor by differential splicing. The different mRNA species are formed by the removal of one to three introns, and they all carry a short 5' leader segment. The introns appear to serve two functions; they either place a 5' leader segment in juxtaposition with an open reading frame or fuse two open translational reading frames. The early region 4 mRNAs can encode at least seven unique polypeptides. Images PMID:6088804

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

    PubMed

    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

    2012-01-18

    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

  13. 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

  14. The mRNAs associated to a zinc finger protein from Trypanosoma cruzi shift during stress conditions

    PubMed Central

    Alves, Lysangela Ronalte; Oliveira, Camila; Mörking, Patrícia Alves; Kessler, Rafael Luis; Martins, Sharon Toledo; Romagnoli, Bruno Accioly Alves; Marchini, Fabricio Kerrynton; Goldenberg, Samuel

    2014-01-01

    Trypanosome gene expression is regulated almost exclusively at the posttranscriptional level, through mRNA stability, storage and degradation. Here, we characterize the ribonucleoprotein complex (mRNPs) corresponding to the zinc finger protein TcZC3H39 from T. cruzi comparing cells growing in normal conditions and under nutritional stress. The nutritional stress is a key step during T. cruzi differentiation from epimastigote form to human infective metacyclic trypomastigote form. The mechanisms by which the stress, altogether with other stimuli, triggers differentiation is not well understood. This work aims to characterize the TcZC3H39 protein during stress response. Using cells cultured in normal and stress conditions, we observed a dynamic change in TcZC3H39 granule distribution, which appeared broader in stressed epimastigotes. The protein core of the TcZC3H39-mRNP is composed of ribosomes, translation factors and RBPs. The TcZC3H39-mRNP could act sequestering highly expressed mRNAs and their associated ribosomes, potentially slowing translation in stress conditions. A shift were observed in the mRNAs associated with TcZC3H39: the number of targets in unstressed epimastigotes was smaller than that in stressed parasites, with no clear functional clustering in normal conditions. By contrast, in stressed parasites, the targets of TcZC3H39 were mRNAs encoding ribosomal proteins and a remarkable enrichment in mRNAs for the cytochrome c complex (COX), highly expressed mRNAs in the replicative form. This identification of a new component of RNA granules in T. cruzi, the TcZC3H39 protein, provides new insight into the mechanisms involved in parasite stress responses and the regulation of gene expression during T. cruzi differentiation. PMID:25180711

  15. 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

  16. RNase-L control of cellular mRNAs: roles in biologic functions and mechanisms of substrate targeting.

    PubMed

    Brennan-Laun, Sarah E; Ezelle, Heather J; Li, Xiao-Ling; Hassel, Bret A

    2014-04-01

    RNase-L is a mediator of type 1 interferon-induced antiviral activity that has diverse and critical cellular roles, including the regulation of cell proliferation, differentiation, senescence and apoptosis, tumorigenesis, and the control of the innate immune response. Although RNase-L was originally shown to mediate the endonucleolytic cleavage of both viral and ribosomal RNAs in response to infection, more recent evidence indicates that RNase-L also functions in the regulation of cellular mRNAs as an important mechanism by which it exerts its diverse biological functions. Despite this growing body of work, many questions remain regarding the roles of mRNAs as RNase-L substrates. This review will survey known and putative mRNA substrates of RNase-L, propose mechanisms by which it may selectively cleave these transcripts, and postulate future clinical applications. PMID:24697205

  17. RNase-L Control of Cellular mRNAs: Roles in Biologic Functions and Mechanisms of Substrate Targeting

    PubMed Central

    Brennan-Laun, Sarah E.; Ezelle, Heather J.; Li, Xiao-Ling

    2014-01-01

    RNase-L is a mediator of type 1 interferon-induced antiviral activity that has diverse and critical cellular roles, including the regulation of cell proliferation, differentiation, senescence and apoptosis, tumorigenesis, and the control of the innate immune response. Although RNase-L was originally shown to mediate the endonucleolytic cleavage of both viral and ribosomal RNAs in response to infection, more recent evidence indicates that RNase-L also functions in the regulation of cellular mRNAs as an important mechanism by which it exerts its diverse biological functions. Despite this growing body of work, many questions remain regarding the roles of mRNAs as RNase-L substrates. This review will survey known and putative mRNA substrates of RNase-L, propose mechanisms by which it may selectively cleave these transcripts, and postulate future clinical applications. PMID:24697205

  18. The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

    PubMed Central

    Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; Fata, Giorgio La; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

    2013-01-01

    The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

  19. The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

    PubMed

    Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; La Fata, Giorgio; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

    2013-10-01

    The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

  20. 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

  1. 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. PMID:25794616

  2. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    PubMed Central

    El-Sayed, Ahmed Kamel; Zhang, Zhentao; Zhang, Lei; Liu, Zhiyong; Abbott, Louise C.; Zhang, Yani; Li, Bichun

    2014-01-01

    Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs) generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF) cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1) and Rex-1 (ZFP-42, zinc finger protein 42). Using embryonic stem cells (ESCs) conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs) using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF) cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations. PMID:25437916

  3. 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

  4. 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.

  5. Isolation of mRNAs associated with yeast mitochondria to study mechanisms of localized translation.

    PubMed

    Lesnik, Chen; Arava, Yoav

    2014-01-01

    Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes' association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors. PMID:24686138

  6. 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.

  7. Discovery of shear- and side-specific mRNAs and miRNAs in human aortic valvular endothelial cells

    PubMed Central

    Holliday, Casey J.; Ankeny, Randall F.; Nerem, Robert M.

    2011-01-01

    The role of endothelial cells (ECs) in aortic valve (AV) disease remains relatively unknown; however, disease preferentially occurs in the fibrosa. We hypothesized oscillatory shear (OS) present on the fibrosa stimulates ECs to modify mRNAs and microRNAs (miRNAs) inducing disease. Our goal was to identify mRNAs and miRNAs differentially regulated by OS and laminar shear (LS) in human AVECs (HAVECs) from the fibrosa (fHAVECs) and ventricularis (vHAVECs). HAVECs expressed EC markers as well as some smooth muscle cell markers and functionally aligned with the flow. HAVECs were exposed to OS and LS for 24 h, and total RNA was analyzed by mRNA and miRNA microarrays. We found over 700 and 300 mRNAs down- and upregulated, respectively, by OS; however, there was no side dependency. mRNA microarray results were validated for 26 of 28 tested genes. Ingenuity Pathway Analysis revealed thrombospondin 1 (Thbs1) and NF-κB inhibitor-α (Nfkbia) as highly connected, shear-sensitive genes. miRNA array analysis yielded 30 shear-sensitive miRNAs and 3 side-specific miRNAs. miRNA validation confirmed 4 of 17 shear-sensitive miRNAs and 1 of 3 side-dependent miRNAs. Using miRWalk and several filtering steps, we identified shear-sensitive mRNAs potentially targeted by shear-sensitive miRNAs. These genes and signaling pathways could act as therapeutic targets of AV disease. PMID:21705672

  8. Overexpression of E2F mRNAs Associated with Gastric Cancer Progression Identified by the Transcription Factor and miRNA Co-Regulatory Network Analysis

    PubMed Central

    Zhang, XiaoTian; Ni, ZhaoHui; Duan, ZiPeng; Xin, ZhuoYuan; Wang, HuaiDong; Tan, JiaYi; Wang, GuoQing; Li, Fan

    2015-01-01

    Gene expression is regulated at the transcription and translation levels; thus, both transcription factors (TFs) and microRNAs (miRNA) play roles in regulation of gene expression. This study profiled differentially expressed mRNAs and miRNAs in gastric cancer tissues to construct a TF and miRNA co-regulatory network in order to identify altered genes in gastric cancer progression. A total of 70 cases gastric cancer and paired adjacent normal tissues were subjected to cDNA and miRNA microarray analyses. We obtained 887 up-regulated and 93 down-regulated genes and 41 down-regulated and 4 up-regulated miRNAs in gastric cancer tissues. Using the Transcriptional Regulatory Element Database, we obtained 105 genes that are regulated by the E2F family of genes and using Targetscan, miRanda, miRDB and miRWalk tools, we predicted potential targeting genes of these 45 miRNAs. We then built up the E2F-related TF and miRNA co-regulatory gene network and identified 9 hub-genes. Furthermore, we found that levels of E2F1, 2, 3, 4, 5, and 7 mRNAs associated with gastric cancer cell invasion capacity, and has associated with tumor differentiation. These data showed Overexpression of E2F mRNAs associated with gastric cancer progression. PMID:25646628

  9. Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic Origin

    PubMed Central

    Lokugamage, Kumari G.; Narayanan, Krishna; Nakagawa, Keisuke; Terasaki, Kaori; Ramirez, Sydney I.; Tseng, Chien-Te K.

    2015-01-01

    ABSTRACT The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome CoV (SARS-CoV) represent highly pathogenic human CoVs that share a property to inhibit host gene expression at the posttranscriptional level. Similar to the nonstructural protein 1 (nsp1) of SARS-CoV that inhibits host gene expression at the translational level, we report that MERS-CoV nsp1 also exhibits a conserved function to negatively regulate host gene expression by inhibiting host mRNA translation and inducing the degradation of host mRNAs. Furthermore, like SARS-CoV nsp1, the mRNA degradation activity of MERS-CoV nsp1, most probably triggered by its ability to induce an endonucleolytic RNA cleavage, was separable from its translation inhibitory function. Despite these functional similarities, MERS-CoV nsp1 used a strikingly different strategy that selectively targeted translationally competent host mRNAs for inhibition. While SARS-CoV nsp1 is localized exclusively in the cytoplasm and binds to the 40S ribosomal subunit to gain access to translating mRNAs, MERS-CoV nsp1 was distributed in both the nucleus and the cytoplasm and did not bind stably to the 40S subunit, suggesting a distinctly different mode of targeting translating mRNAs. Interestingly, consistent with this notion, MERS-CoV nsp1 selectively targeted mRNAs, which are transcribed in the nucleus and transported to the cytoplasm, for translation inhibition and mRNA degradation but spared exogenous mRNAs introduced directly into the cytoplasm or virus-like mRNAs that originate in the cytoplasm. Collectively, these data point toward a novel viral strategy wherein the cytoplasmic origin of MERS-CoV mRNAs facilitates their escape from the inhibitory effects of MERS-CoV nsp1. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human CoV that emerged in Saudi Arabia in 2012. MERS-CoV has a zoonotic origin and poses a major threat to public health

  10. Aryl Hydrocarbon Receptor-Dependent Induction of Flavin-Containing Monooxygenase mRNAs in Mouse LiverS

    PubMed Central

    Celius, Trine; Roblin, Steven; Harper, Patricia A.; Matthews, Jason; Boutros, Paul C.; Pohjanvirta, Raimo; Okey, Allan B.

    2016-01-01

    Flavin-containing monooxygenases (FMOs) are important in detoxication but generally are considered not to be inducible by xenobiotics. Our recent microarray studies revealed induction of FMO2 and FMO3 mRNAs by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver of mice with wild-type aryl hydrocarbon receptor (AHR) but not in Ahr-null mice. The aim of the present study was to delineate mechanisms of FMO regulation. In adult male mice, basal FMO3 mRNA is low but was induced 6-fold at 4 h and 6000-fold at 24 h. The ED50 was approximately 1 μg/kg for FMO2 and FMO3, similar to that for the classic AHR-regulated gene, Cyp1a1. In adult female mice basal FMO3 mRNA is high and was not induced at 4 h but was elevated 8-fold at 24 h. FMO5 mRNA was significantly down-regulated by TCDD in both male and female adult mice. Juvenile mice show no sex difference in response to TCDD; FMO3 was induced 4 to 6-fold by TCDD in both sexes. Chromatin immuno-precipitation demonstrated recruitment of AHR and aryl hydrocarbon nuclear translocator proteins to Fmo3 regulatory regions, suggesting that induction by TCDD is a primary AHR-mediated event. Although FMO2 and FMO3 mRNAs were highly induced by TCDD in adult males, overall FMO catalytic activity increased only modestly. In contrast to the striking up-regulation of FMO2 and FMO3 in mouse liver, TCDD has little effect on FMO mRNA in rat liver. However, FMO2 and FMO3 mRNAs were highly induced in transgenic mice that express wild-type rat AHR, indicating that lack of induction in rat is not due to an incompetent AHR in this species. PMID:18765683

  11. 5'-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation.

    PubMed

    Beck, Heather J; Fleming, Ian M C; Janssen, Gary R

    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

  12. 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. PMID:26668315

  13. 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

  14. Analysis of Nitric Oxide-Stabilized mRNAs in Human Fibroblasts Reveals HuR-Dependent Heme Oxygenase 1 Upregulation▿ †

    PubMed Central

    Kuwano, Yuki; Rabinovic, Ariel; Srikantan, Subramanya; Gorospe, Myriam; Demple, Bruce

    2009-01-01

    We previously observed that nitric oxide (NO) exposure increases the stability of mRNAs encoding heme oxygenase 1 (HO-1) and TIEG-1 in human and mouse fibroblasts. Here, we have used microarrays to look broadly for changes in mRNA stability in response to NO treatment. Using human IMR-90 and mouse NIH 3T3 fibroblasts treated with actinomycin D to block de novo transcription, microarray analysis suggested that the stability of the majority of mRNAs was unaffected. Among the mRNAs that were stabilized by NO treatment, seven transcripts were found in both IMR-90 and NIH 3T3 cells (CHIC2, GADD45B, HO-1, PTGS2, RGS2, TIEG, and ID3) and were chosen for further analysis. All seven mRNAs showed at least one hit of a signature motif for the stabilizing RNA-binding protein (RBP) HuR; accordingly, ribonucleoprotein immunoprecipitation analysis revealed that all seven mRNAs associated with HuR. In keeping with a functional role of HuR in the response to NO, a measurable fraction of HuR increased in the cytoplasm following NO treatment. However, among the seven transcripts, only HO-1 mRNA showed a robust increase in the level of its association with HuR following NO treatment. In turn, HO-1 mRNA and protein levels were significantly reduced when HuR levels were silenced in IMR-90 cells, and they were elevated when HuR was overexpressed. In sum, our results indicate that NO stabilizes mRNA subsets in fibroblasts, identify HuR as an RBP implicated in the NO response, reveal that HuR alone is insufficient for stabilizing several mRNAs by NO, and show that HO-1 induction by NO is regulated by HuR. PMID:19289500

  15. Alternative mechanisms of initiating translation of mammalian mRNAs.

    PubMed

    Jackson, R J

    2005-12-01

    Of all the steps in mRNA translation, initiation is the one that differs most radically between prokaryotes and eukaryotes. Not only is there no equivalent of the prokaryotic Shine-Dalgarno rRNA-mRNA interaction, but also what requires only three initiation factor proteins (aggregate size approximately 125 kDa) in eubacteria needs at least 28 different polypeptides (aggregate >1600 kDa) in mammalian cells, which is actually larger than the size of the 40 S ribosomal subunit. Translation of the overwhelming majority of mammalian mRNAs occurs by a scanning mechanism, in which the 40 S ribosomal subunit, primed for initiation by the binding of several initiation factors including the eIF2 (eukaryotic initiation factor 2)-GTP-MettRNA(i) complex, is loaded on the mRNA immediately downstream of the 5'-cap, and then scans the RNA in the 5'-->3' direction. On recognition of (usually) the first AUG triplet via base-pairing with the Met-tRNA(i) anticodon, scanning ceases, triggering GTP hydrolysis and release of eIF2-GDP. Finally, ribosomal subunit joining and the release of the other initiation factors completes the initiation process. This sketchy outline conceals the fact that the exact mechanism of scanning and the precise roles of the initiation factors remain enigmatic. However, the factor requirements for initiation site selection on some viral IRESs (internal ribosome entry sites/segments) are simpler, and investigations into these IRES-dependent mechanisms (particularly picornavirus, hepatitis C virus and insect dicistrovirus IRESs) have significantly enhanced our understanding of the standard scanning mechanism. This article surveys the various alternative mechanisms of initiation site selection on mammalian (and other eukaryotic) cellular and viral mRNAs, starting from the simplest (in terms of initiation factor requirements) and working towards the most complex, which paradoxically happens to be the reverse order of their discovery. PMID:16246087

  16. 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. PMID:24367277

  17. 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

  18. Integrated analysis of noncoding RNAs and mRNAs reveals their potential roles in the biological activities of the growth hormone receptor.

    PubMed

    Chang, Lei; Qi, Haolong; Xiao, Yusha; Li, Changsheng; Wang, Yitao; Guo, Tao; Liu, Zhisu; Liu, Quanyan

    2016-08-01

    Accumulating evidence has indicated that noncoding RNAs (ncRNAs) have important regulatory potential in various biological processes. The molecular mechanisms by which growth hormone receptor (GHR) deficiency protects against age-related pathologies, reduces the incidence and delays the occurrence of fatal neoplasms are unclear. The aim of this study was to investigate miRNA, lncRNA and mRNA expression profiles and the potential functional roles of these RNA molecules in GHR knockout (GHR-KO) mice. Microarray expression profiles of miRNAs, lncRNAs and mRNAs were determined in wild type control mice and in GHR-KO mice. Differential expression, pathway and gene network analyses were developed to identify the possible biological roles of functional RNA molecules. Compared to wild type control mice, 1695 lncRNAs, 914 mRNAs and 9 miRNAs were upregulated and 1747 lncRNAs, 786 mRNAs and 21 miRNAs were downregulated in female GHR-KO mice. Moreover, 1265 lncRNAs, 724 mRNAs and 41 miRNAs were upregulated and 1377 lncRNAs, 765 mRNAs and 16 miRNAs were downregulated in male GHR-KO mice compared to wild type mice. Co-expression analysis of mRNAs, lncRNAs, and miRNAs showed that mRNAs including Hemxi2, Ero1Ib, 4933434i20RIK, Pde7a and Lgals1, lncRNAs including ASMM9PARTA014848, EL605414-P1, ASMM9PARTA051724, ASMM9PARTA045378 and ASMM9PARTA049185, and miRNAs including miR-188-3p, miR-690, miR-709 and miR-710 are situated at the core position of a three-dimensional lncRNA-mRNA-miRNA regulatory network. KEGG analysis showed that the most significantly regulated pathway was steroid hormone biosynthesis. We identified a set of lncRNAs, miRNAs and mRNAs that were aberrantly expressed in GHR-KO mice. Our results provide a foundation and an expansive view of the biological activities of the GHR. PMID:27064376

  19. Role of 5'- and 3'-untranslated regions of mRNAs in human diseases.

    PubMed

    Chatterjee, Sangeeta; Pal, Jayanta K

    2009-05-01

    Protein synthesis is often regulated at the level of initiation of translation, making it a critical step. This regulation occurs by both the cis-regulatory elements, which are located in the 5'- and 3'-UTRs (untranslated regions), and trans-acting factors. A breakdown in this regulation machinery can perturb cellular metabolism, leading to various physiological abnormalities. The highly structured UTRs, along with features such as GC-richness, upstream open reading frames and internal ribosome entry sites, significantly influence the rate of translation of mRNAs. In this review, we discuss how changes in the cis-regulatory sequences of the UTRs, for example, point mutations and truncations, influence expression of specific genes at the level of translation. Such modifications may tilt the physiological balance from healthy to diseased states, resulting in conditions such as hereditary thrombocythaemia, breast cancer, fragile X syndrome, bipolar affective disorder and Alzheimer's disease. This information tends to establish the crucial role of UTRs, perhaps as much as that of coding sequences, in health and disease. PMID:19275763

  20. The histone H3 and H4 mRNAs are polyadenylated in maize.

    PubMed Central

    Chaubet, N; Chaboute, M E; Clément, B; Ehling, M; Philipps, G; Gigot, C

    1988-01-01

    Northern blot analysis revealed that the histone H3 and H4 mRNAs are of unusual large size in germinating maize embryos. S1-mapping experiments show that the 3'-untranslated regions of the mRNAs transcribed from 3 H3 and 2 H4 maize genes previously described are much longer than in the non-polyadenylated histone mRNAs which represent a major class in animals. Moreover, oligo d(T) cellulose fractionation of RNAs isolated at different developmental stages indicates that more than 99% of the maize H3 and H4 mRNAs are polyadenylated. A putative polyadenylation signal is present in all five genes 17 to 27 nucleotides before the 3'-ends of the mRNAs. Images PMID:2831497

  1. 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

  2. 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

  3. 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. PMID:21737422

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

    PubMed Central

    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

    2011-01-01

    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. PMID:21737422

  5. Interplay of viral miRNAs and host mRNAs and proteins

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir

    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.

  6. Dysregulated expression of microRNAs and mRNAs in myocardial infarction

    PubMed Central

    Wang, Yaping; Pan, Xiaohong; Fan, Youqi; Hu, Xinyang; Liu, Xianbao; Xiang, Meixiang; Wang, Jian’an

    2015-01-01

    Acute myocardial infarction (AMI) is a major cause of mortality in the general population. However, the molecular phenotypes and therapeutic targets of AMI patients remain unclear. By profiling genome-wide transcripts and microRNAs (miRNAs) in a cohort of 23 AMI patients and 23 non-AMI patients, we found 218 dysregulated genes identified in the infarcted heart tissues from AMI patients relative to non-AMI controls. Pathway enrichment analysis of the dysregulated genes pointed to cell signaling/communication, cell/organism defense and cell structure/motility. We next compared the expression profiles of potential regulating miRNAs, suggesting that dysregulation of a number of AMI-associated genes (e.g., IL12A, KIF1A, HIF1α and CDK13) may be attributed to the dysregulation of their respective regulating miRNAs. One potentially pathogenic miRNA-mRNA pair, miR-210-HIF1α, was confirmed in a mouse model of myocardial infarction (MI). Inhibition of miR-210 expression improved the survival and cardiac function of MI mice. In conclusion, we presented the pathologic relationships between miRNAs and their gene targets in AMI. Such deregulated microRNAs and mRNAs like miR-210 serve as novel therapeutic targets of AMI. PMID:26807177

  7. Bicaudal-C spatially controls translation of vertebrate maternal mRNAs

    PubMed Central

    Zhang, Yan; Cooke, Amy; Park, Sookhee; Dewey, Colin N.; Wickens, Marvin; Sheets, Michael D.

    2013-01-01

    The Xenopus Cripto-1 protein is confined to the cells of the animal hemisphere during early embryogenesis where it regulates the formation of anterior structures. Cripto-1 protein accumulates only in animal cells because cripto-1 mRNA in cells of the vegetal hemisphere is translationally repressed. Here, we show that the RNA binding protein, Bicaudal-C (Bic-C), functioned directly in this vegetal cell-specific repression. While Bic-C protein is normally confined to vegetal cells, ectopic expression of Bic-C in animal cells repressed a cripto-1 mRNA reporter and associated with endogenous cripto-1 mRNA. Repression by Bic-C required its N-terminal domain, comprised of multiple KH motifs, for specific binding to relevant control elements within the cripto-1 mRNA and a functionally separable C-terminal translation repression domain. Bic-C-mediated repression required the 5′ CAP and translation initiation factors, but not a poly(A) tail or the conserved SAM domain within Bic-C. Bic-C-directed immunoprecipitation followed by deep sequencing of associated mRNAs identified multiple Bic-C-regulated mRNA targets, including cripto-1 mRNA, providing new insights and tools for understanding the role of Bic-C in vertebrate development. PMID:24062572

  8. The differential expression of mRNAs and long noncoding RNAs between ectopic and eutopic endometria provides new insights into adenomyosis.

    PubMed

    Zhou, Cheng; Zhang, Ting; Liu, Fei; Zhou, Ji; Ni, Xiaobei; Huo, Ran; Shi, Zhonghua

    2016-02-01

    Adenomyosis, defined as ectopic endometrial tissue within the myometrium, can often be misdiagnosed as multiple uterine leiomyomata or endometrial thickening. We therefore performed a combined mRNA and long noncoding (lnc)RNA microarray and bioinformatic analysis of eutopic and ectopic endometria in women with adenomyosis to better understand its pathogenesis and help in the development of a semi-invasive diagnostic test. A total of 586 mRNAs were increased and 305 mRNAs decreased in the ectopic endometrium of adenomyosis compared with the eutopic endometrium, while 388 lncRNA transcripts were up-regulated and 188 down-regulated in ectopic compared with paired eutopic endometrial tissue. Bioinformatic analysis suggested a series of metabolic and molecular abnormalities in adenomyosis, which have many similarities with endometriosis. Furthermore, our study constitutes the first known report of lncRNA expression patterns in human adenomyosis ectopic and eutopic endometrial tissue. PMID:26662114

  9. Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae.

    PubMed Central

    Herrick, D; Parker, R; Jacobson, A

    1990-01-01

    We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs. Images PMID:2183028

  10. Levels of mRNAs which code for small, acid-soluble spore proteins and their LacZ gene fusions in sporulating cells of Bacillus subtilis.

    PubMed Central

    Mason, J M; Fajardo-Cavazos, P; Setlow, P

    1988-01-01

    The levels of mRNAs from genes (sspA, B and E) which code for major small, acid-soluble, spore proteins of Bacillus subtilis have been determined, as well as the levels of mRNAs from ssp-lacZ gene fusions. Increasing the gene dosage of ssp-lacZ fusions resulted in parallel increases in both the ssp-lacZ mRNA level and the rate of b-galactosidase accumulation. Similarly, an 11-fold increase in sspE gene dosage gave a comparable increase in sspE mRNA, but at most a 1.5-fold increase in the amount of sspE gene product accumulated. In contrast, an 11-fold increase in the dosage of the sspA or B genes had no significant effect on the level of total sspA plus sspB mRNA, but did alter the ratios of these mRNAs as well as the amount of their gene products, to reflect the altered ratio of the two genes. These results suggest that intact ssp genes, but not ssp-lacZ gene fusions, are subject to feedback regulation of gene expression, with this regulation of the sspA and B genes effected by modulation of mRNA levels, while the feedback regulation of the sspE gene is at the post-transcriptional level. Images PMID:2456528

  11. Very short-lived and stable mRNAs from resting human lymphocytes.

    PubMed Central

    Berger, S L; Cooper, H L

    1975-01-01

    The kinetics of degradation of newly synthesized cytoplasmic poly(A)-bearing RNA have been examined in resting human lymphocytes. Two classes were identified, a very labile component with a half-life of less than 17 min and a stable component which remains apparently undiminished during 24 hr of observation. Both classes have molecular weights between 2.5 and 3.5 x 10(5) but the stable material has a narrower size distribution and a slightly lower average molecular weight than the short-lived component. The fate of stable RNA synthesized in the resting cell was also examined after growth stimulation with phytohemagglutinin after 2 and 6 hr of treatment. No transfer of stable material into the labile pool could be discerned; the amount of stable material remained constant. The existence of two species of mRNAs with different lifetimes in animal cells provides a potential means for regulation of protein synthesis by controlling the supply of specific messages. Furthermore, such a short-lived mRNA species may explain the observed disparity between the amount of poly(A)-bearing heterogeneous RNA produced in the nucleus and the amount of mature message found in the cytoplasm. PMID:1060069

  12. 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

  13. Distal Alternative Last Exons Localize mRNAs to Neural Projections.

    PubMed

    Taliaferro, J Matthew; Vidaki, Marina; Oliveira, Ruan; Olson, Sara; Zhan, Lijun; Saxena, Tanvi; Wang, Eric T; Graveley, Brenton R; Gertler, Frank B; Swanson, Maurice S; Burge, Christopher B

    2016-03-17

    Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer. PMID:26907613

  14. 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.

  15. Differential stability of c-myc mRNAS in a cell-free system

    SciTech Connect

    Pei, R.; Calame, K.

    1988-07-01

    The authors have developed a simple cell-free system for studying the stability of different mRNAs in vitro. They demonstrate that the threefold greater stability in vivo of truncated c-myc mRNA (lacking exon 1) compared with that of full-length c-myc mRNA is maintained in our in vitro system. Chimeric mRNAs in which the first exon of c-myc was fused to immunoglobulin C ..cap alpha.. heavy chain of glyceraldehyde-3-phosphate dehydrogenase mRNAs were not rapidly degraded, demonstrating that c-myc exon 1 alone is not sufficient to tag mRNAs for rapid degradation. Competition experiments show that full-length c-myc mRNA is specifically recognized by a factor(s) responsible for its rapid degradation. This system will allow further characterization and purification of these factors.

  16. Expression of mRNAs encoding mammalian chromosomal proteins HMG-I and HMG-Y during cellular proliferation

    SciTech Connect

    Johnson, K.R.; Disney, J.E.; Wyatt, C.R.; Reeves, R. )

    1990-03-01

    The high mobility group chromosomal proteins HMG-I and HMG-Y are closely related isoforms that are expressed at high levels in rapidly dividing, undifferentiated mammalian cells. The authors analyzed HMG-I/Y mRNA levels at various cell cycle stages in murine NIH/3T3 fibroblasts partially synchronized by seeding from quiescent, contact-inhibited cultures. Flow microfluorometric analysis of DNA content demonstrated a comparable degree of synchronization in such seeded NIH 3T3 cell populations as is obtained by serum deprivation or other means and has the added advantage of avoiding the use of possibly detrimental inhibitors or metabolic starvation to induce such synchrony. They show that HMG-I/Y mRNA levels gradually increase in NIH/3T3 cells during the first 16 hours after seeding (G{sub 0}/G{sub 1} to late S phase), but thereafter remain constant, in contrast to the cell cycle-regulated expression of the histone H3 gene. The HMG-I/Y mRNAs appear to be very stable; there was no decrease in their levels 6 hours after actinomycin D transcription termination. The proportion of HMG-I to HMG-Y mRNAs was greater in the human than in the murine cells examined, appeared to be greater in proliferating than in quiescent cells, and did not always correspond with the HMG-I to HMG-Y protein ratio.

  17. 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.

  18. 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

  19. IGF2BP2/IMP2-Deficient mice resist obesity through enhanced translation of Ucp1 mRNA and Other mRNAs encoding mitochondrial proteins.

    PubMed

    Dai, Ning; Zhao, Liping; Wrighting, Diedra; Krämer, Dana; Majithia, Amit; Wang, Yanqun; Cracan, Valentin; Borges-Rivera, Diego; Mootha, Vamsi K; Nahrendorf, Matthias; Thorburn, David R; Minichiello, Liliana; Altshuler, David; Avruch, Joseph

    2015-04-01

    Although variants in the IGF2BP2/IMP2 gene confer risk for type 2 diabetes, IMP2, an RNA binding protein, is not known to regulate metabolism. Imp2(-/-) mice gain less lean mass after weaning and have increased lifespan. Imp2(-/-) mice are highly resistant to diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and better defense of core temperature on cold exposure. Imp2(-/-) brown fat and Imp2(-/-) brown adipocytes differentiated in vitro contain more UCP1 polypeptide than Imp2(+/+) despite similar levels of Ucp1 mRNA; the Imp2(-/-)adipocytes also exhibit greater uncoupled oxygen consumption. IMP2 binds the mRNAs encoding Ucp1 and other mitochondrial components, and most exhibit increased translational efficiency in the absence of IMP2. In vitro IMP2 inhibits translation of mRNAs bearing the Ucp1 untranslated segments. Thus IMP2 limits longevity and regulates nutrient and energy metabolism in the mouse by controlling the translation of its client mRNAs. PMID:25863250

  20. Brain-expressed 3′UTR extensions strengthen miRNA cross-talk between ion channel/transporter encoding mRNAs

    PubMed Central

    Wehrspaun, Claudia C.; Ponting, Chris P.; Marques, Ana C.

    2014-01-01

    Why protein-coding genes express transcripts with longer 3′untranslated regions (3′UTRs) in the brain rather than in other tissues remains poorly understood. Given the established role of 3′UTRs in post-transcriptional regulation of transcript abundance and their recently highlighted contributions to miRNA-mediated cross-talk between mRNAs, we hypothesized that 3′UTR lengthening enhances coordinated expression between functionally-related genes in the brain. To test this hypothesis, we annotated 3′UTRs of human brain-expressed genes and found that transcripts encoding ion channels or transporters are specifically enriched among those genes expressing their longest 3′UTR extension in this tissue. These 3′UTR extensions have high density of response elements predicted for those miRNAs that are specifically expressed in the human frontal cortex (FC). Importantly, these miRNA response elements are more frequently shared among ion channel/transporter-encoding mRNAs than expected by chance. This indicates that miRNA-mediated cross-talk accounts, at least in part, for the observed coordinated expression of ion channel/transporter genes in the adult human brain. We conclude that extension of these genes' 3′UTRs enhances the miRNA-mediated cross-talk among their transcripts which post-transcriptionally regulates their mRNAs' relative levels. PMID:24616735

  1. Expression of hepatic mRNAs for insulin-like growth factors-I and -II during the development of hypothyroid rats.

    PubMed

    Gallo, G; de Marchis, M; Voci, A; Fugassa, E

    1991-12-01

    The effect of thyroid status on the expression of insulin-like growth factors-I and -II mRNAs in the liver of developing rats has been investigated. Northern blot analyses of the specific mRNA demonstrated the presence of four IGF-II mRNA species which were strongly expressed in fetal liver and progressively declined after birth, becoming undetectable after week 3. This decrease was markedly delayed in the liver of hypothyroid rats. In addition, expression of IGF-I mRNA, absent in fetal liver, began during week 1 after birth and progressively increased with age. This increase was markedly delayed in the liver of hypothyroid rats. The data suggest that thyroid hormones regulate rat development via the co-ordinate expression of hepatic IGF-II and IGF-I mRNAs. PMID:1783883

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

    PubMed

    García-Alcocer, G; García-Colunga, J; Martínez-Torres, A; Miledi, R

    2001-02-27

    A study was made of glycine (Gly) and gamma-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 < or =40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC(50) for Gly was 611 +/- 77 microM for encephalon, 661 +/- 28 microM for midbrain, and 506 +/- 18 microM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all approximately 2. Strychnine inhibited all of the Gly-currents, with an IC(50) 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 Ca(2+) 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) alpha-subunits expressed, whereas the beta-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 Ca(2+) could play important functions during brain development. PMID:11226317

  3. A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

    PubMed Central

    Eystathioy, Theophany; Chan, Edward K. L.; Tenenbaum, Scott A.; Keene, Jack D.; Griffith, Kevin; Fritzler, Marvin J.

    2002-01-01

    A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis. PMID:11950943

  4. Differential nonsense mediated decay of mutated mRNAs in mismatch repair deficient colorectal cancers.

    PubMed

    El-Bchiri, Jamila; Buhard, Olivier; Penard-Lacronique, Virginie; Thomas, Gilles; Hamelin, Richard; Duval, Alex

    2005-08-15

    The nonsense-mediated decay (NMD) system normally targets mRNAs with premature termination codons (PTCs) for rapid degradation. We investigated for a putative role of NMD in cancers with microsatellite instability (MSI-H cancers), because numerous mutant mRNAs containing PTC are generated in these tumors as a consequence of their mismatch repair deficiency. Using a quantitative RT-PCR approach in a large series of colorectal cancer cell lines, we demonstrate a significantly increased rate of degradation of mutant mRNAs containing a PTC compared with wild-type. A specific siRNA strategy was used to inhibit RENT-1 and/or RENT-2 activity, two major genes in the NMD system. This allowed us to show that increased degradation of PTC-containing mRNAs in MSI-H tumors was partly dependent upon NMD activity. The efficiency of NMD for the degradation of mutant mRNAs from target genes was highly variable in these cancers. NMD degraded some of them (TGFssRII, MSH3, GRK4), although allowing the persistent expression of others (BAX, TCF-4). This is of particular interest within the context of a proposed conservation of biological activity for the corresponding mutated proteins. We thus propose that NMD might play an important role in the selection of target gene mutations with a functional role in MSI-H carcinogenesis. PMID:16000315

  5. 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.

  6. 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

  7. 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. PMID:26762974

  8. UTRdb and UTRsite: specialized databases of sequences and functional elements of 5′ and 3′ untranslated regions of eukaryotic mRNAs

    PubMed Central

    Pesole, Graziano; Liuni, Sabino; Grillo, Giorgio; Licciulli, Flavio; Larizza, Alessandra; Makalowski, Wojciech; Saccone, Cecilia

    2000-01-01

    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, 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. All internet resources implemented for retrieval and functional analysis of 5′ and 3′ untranslated regions of eukaryotic mRNAs are accessible at http://bigarea.area.ba.cnr.it:8000/EmbIT/UTRHome/ PMID:10592223

  9. UTRdb and UTRsite: specialized databases of sequences and functional elements of 5′ and 3′ untranslated regions of eukaryotic mRNAs. Update 2002

    PubMed Central

    Pesole, Graziano; Liuni, Sabino; Grillo, Giorgio; Licciulli, Flavio; Mignone, Flavio; Gissi, Carmela; Saccone, Cecilia

    2002-01-01

    The 5′- and 3′-untranslated regions (5′- and 3′-UTRs) of eukaryotic mRNAs are known to play a crucial role in post-transcriptional regulation of gene expression modulating nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and stability. UTRdb is 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. All Internet resources we implemented for retrieval and functional analysis of 5′- and 3′-UTRs of eukaryotic mRNAs are accessible at http://bighost.area.ba.cnr.it/BIG/UTRHome/. PMID:11752330

  10. 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.

  11. Translation of a Small Subset of Caenorhabditis elegans mRNAs Is Dependent on a Specific Eukaryotic Translation Initiation Factor 4E Isoform

    PubMed Central

    Dinkova, Tzvetanka D.; Keiper, Brett D.; Korneeva, Nadejda L.; Aamodt, Eric J.; Rhoads, Robert E.

    2005-01-01

    The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E. PMID:15601834

  12. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    PubMed

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. PMID:27167364

  13. Rapid degradation of replication-dependent histone mRNAs largely occurs on mRNAs bound by nuclear cap-binding proteins 80 and 20

    PubMed Central

    Choe, Junho; Kim, Kyoung Mi; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Kim, Min Kyung; Lee, Byung-Gil; Song, Hyun Kyu; Kim, Yoon Ki

    2013-01-01

    The translation of mammalian messenger RNAs (mRNAs) can be driven by either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF)4E. Although CBP80/20-dependent translation (CT) is known to be coupled to an mRNA surveillance mechanism termed nonsense-mediated mRNA decay (NMD), its molecular mechanism and biological role remain obscure. Here, using a yeast two-hybrid screening system, we identify a stem-loop binding protein (SLBP) that binds to a stem-loop structure at the 3′-end of the replication-dependent histone mRNA as a CT initiation factor (CTIF)-interacting protein. SLBP preferentially associates with the CT complex of histone mRNAs, but not with the eIF4E-depedent translation (ET) complex. Several lines of evidence indicate that rapid degradation of histone mRNA on the inhibition of DNA replication largely takes place during CT and not ET, which has been previously unappreciated. Furthermore, the ratio of CBP80/20-bound histone mRNA to eIF4E-bound histone mRNA is larger than the ratio of CBP80/20-bound polyadenylated β-actin or eEF2 mRNA to eIF4E-bound polyadenylated β-actin or eEF2 mRNA, respectively. The collective findings suggest that mRNAs harboring a different 3′-end use a different mechanism of translation initiation, expanding the repertoire of CT as a step for determining the fate of histone mRNAs. PMID:23234701

  14. Unequal distribution of N6-methyladenosine in influenza virus mRNAs.

    PubMed

    Narayan, P; Ayers, D F; Rottman, F M; Maroney, P A; Nilsen, T W

    1987-04-01

    Influenza virus mRNA is posttranscriptionally methylated at internal adenosine residues to form N6-methyladenosine (m6A). It has been previously shown that there is an average of three m6A residues per influenza virus mRNA (R. M. Krug, M. A. Morgan, and A. J. Shatkin, J. Virol. 20:45-53, 1976). To determine the distribution of m6A in the different influenza virus mRNAs, we purified six of the mRNAs by hybrid selection, digested them with nuclease, and determined their methylation patterns by high-pressure liquid chromatography. The amount of m6A in the different mRNAs varied from one in matrix to eight in hemagglutinin. PMID:3600638

  15. Developmental changes in translatable mRNAs for the cerebral enolase isozymes alphaalpha and gammagamma.

    PubMed Central

    Zeitoun, Y; Lamandé, N; Keller, A; Gros, F; Legault-Demare, L

    1983-01-01

    Using the rabbit reticulocyte cell-free translation system we have estimated during ontogenesis the proportions of in vitro translatable alpha and gamma brain enolase mRNAs, which are two minor mRNA species. No polypeptide precursor to these enzyme subunits appears to be synthesized during translation in vitro. During brain development, the changes in translatable alpha and gamma mRNA content seem to parallel those of the corresponding antigens. The proportion of each of the enolase mRNAs is highest in adult mouse brain. Mechanisms controlling alpha and gamma antigen expression are discussed. In order to prepare the specific cDNA probes, purification of alpha and gamma mRNAs was undertaken. Images Fig. 1. Fig. 2. Fig. 4. PMID:11892794

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

    PubMed Central

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

    2016-01-01

    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. PMID:27313041

  17. 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. PMID:27276446

  18. 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. PMID:27313041

  19. 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

  20. 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. PMID:23827238

  1. Organ-Specific Stability of Two Lemna rbcS mRNAs Is Determined Primarily in the Nuclear Compartment.

    PubMed Central

    Peters, J. L.; Silverthorne, J.

    1995-01-01

    It has previously been shown that the organ-specific expression of two members of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene family is post-transcriptionally regulated in Lemna gibba. While both small subunit genes encoding SSU1 and SSU5B were transcribed at comparable levels in root and frond nuclei, SSU1 mRNA accumulated to high levels in both roots and fronds in contrast to SSU5B mRNA, which was of very low abundance in the roots compared with the fronds. In this study, we have used two approaches to pinpoint the step(s) at which SSU1 and SSU5B mRNAs are differentially accumulated in these organs. In the first approach, total nuclear steady state mRNA was isolated from roots and fronds, and the amount of each transcript was measured by RNase protection assays and compared with the transcription rates in isolated nuclei. In the second approach, cordycepin was used to inhibit mRNA synthesis in Lemna fronds or roots, and the rate of decay of each mRNA was measured by RNA gel blot analysis or RNase protection assays. Our findings indicate that the differential accumulation of SSU1 and SSU5B mRNAs in the fronds versus the roots is determined primarily in the nuclear compartment. In addition, SSU1 was found to have a longer half-life in total steady state mRNA than SSU5B had in both organs. This feature probably accounts for SSU1 being the predominantly expressed family member. PMID:12242353

  2. Embryonic Corneal Schwann Cells Express Some Schwann Cell Marker mRNAs, but No Mature Schwann Cell Marker Proteins

    PubMed Central

    Conrad, Abigail H.; Albrecht, Michael; Pettit-Scott, Maya; Conrad, Gary W.

    2009-01-01

    Purpose Embryonic chick nerves encircle the cornea in pericorneal tissue until embryonic day (E)9, then penetrate the anterior corneal stroma, invade the epithelium, and branch over the corneal surface through E20. Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas. Methods Fertile chicken eggs were incubated from E0 at 38°C, 45% humidity. Dissected permeabilized corneas plus pericorneal tissue were immunostained for SC marker proteins. Other corneas were paraffin embedded, sectioned, and processed by in situ hybridization for corneal-, nerve-related, and SC marker gene expression. E9 to E20 corneas, dissected from pericorneal tissue, were assessed by real-time PCR (QPCR) for mRNA expression. Results QPCR revealed unchanging low to moderate SLIT2/ROBO and NTN/UNC5 family, BACE1, and CADM3/CADM4 expressions, but high NEO1 expression. EGR2 and POU3F1 expressions never surpassed PAX3 expression. ITGNA6/IT-GNB4 expressions increased 20-fold; ITGNB1 expression was high. SC marker S100 and MBP expressions increased; MAG, GFAP, and SCMP expressions were very low. Antibodies against the MPZ, MAG, S100, and SCMP proteins immunostained along pericorneal nerves, but not along corneal nerves. In the cornea, SLIT2 and SOX10 mRNAs were expressed in anterior stroma and epithelium, whereas PAX3, S100, MBP, and MPZL1 mRNAs were expressed only in corneal epithelium. Conclusions Embryonic chick corneas contain SCs, as defined by SOX10 and PAX3 transcription, which remain immature, at least in part because of stromal transcriptional and epithelial translational regulation of some SC marker gene expression. PMID:19387082

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

    PubMed Central

    2013-01-01

    Background 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. Results 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. Conclusions 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. PMID:23915301

  4. 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. PMID:26881311

  5. A profusion of upstream open reading frame mechanisms in polyamine-responsive translational regulation.

    PubMed

    Ivanov, Ivaylo P; Atkins, John F; Michael, Antony J

    2010-01-01

    In many eukaryotic mRNAs one or more short 'upstream' open reading frames, uORFs, precede the initiator of the main coding sequence. Upstream ORFs are functionally diverse as illustrated by their variety of features in polyamine pathway biosynthetic mRNAs. Their propensity to act as sensors for regulatory circuits and to amplify the signals likely explains their occurrence in most polyamine pathway mRNAs. The uORF-mediated polyamine responsive autoregulatory circuits found in polyamine pathway mRNAs exemplify the translationally regulated dynamic interface between components of the proteome and metabolism. PMID:19920120

  6. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells.

    PubMed

    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

    2008-05-23

    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

  7. Ribonuclease III-mediated processing of specific Neisseria meningitidis mRNAs.

    PubMed Central

    De Gregorio, Eliana; Abrescia, Chiara; Carlomagno, M Stella; Di Nocera, Pier Paolo

    2003-01-01

    Approx. 2% of the Neisseria meningitidis genome consists of small DNA insertion sequences known as Correia or nemis elements, which feature TIRs (terminal inverted repeats) of 26-27 bp in length. Elements interspersed with coding regions are co-transcribed with flanking genes into mRNAs, processed at double-stranded RNA structures formed by TIRs. N. meningitidis RNase III (endoribonuclease III) is sufficient to process nemis+ RNAs. RNA hairpins formed by nemis with the same termini (26/26 and 27/27 repeats) are cleaved. By contrast, bulged hairpins formed by 26/27 repeats inhibit cleavage, both in vitro and in vivo. In electrophoretic mobility shift assays, all hairpin types formed similar retarded complexes upon incubation with RNase III. The levels of corresponding nemis+ and nemis- mRNAs, and the relative stabilities of RNA segments processed from nemis+ transcripts in vitro, may both vary significantly. PMID:12826014

  8. Spatial and temporal accumulation of mRNAs encoding two common lignin peroxidases in Phanerochaete chrysosporium.

    PubMed Central

    Moukha, S M; Wösten, H A; Mylius, E J; Asther, M; Wessels, J G

    1993-01-01

    Accumulation of peroxidases and their mRNAs was localized in colonies of Phanerochaete chrysosporium sandwiched between perforated polycarbonate membranes. Northern (RNA) blot analyses of colonial rings and in situ hybridizations with specific probes for manganese(II)-dependent peroxidase (MnP-1) and lignin peroxidase (LiP H8) mRNAs indicated that the expression of MnP-1 and Lip H8 genes started simultaneously in the central area of 3-day-old colonies. With time the signals for both transcripts spread to more-peripheral areas while decreasing in intensity. Furthermore, the appearance of MnP protein, as detected with specific immune serum, immediately followed accumulation of the MnP-1 mRNA transcript. However, LiP protein could be detected only some time after accumulation of LiP H8 mRNA. Images PMID:8501073

  9. 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

  10. Death of a dogma: eukaryotic mRNAs can code for more than one protein.

    PubMed

    Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

    2016-01-01

    mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. PMID:26578573

  11. Death of a dogma: eukaryotic mRNAs can code for more than one protein

    PubMed Central

    Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

    2016-01-01

    mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5′ UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3′ UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. PMID:26578573

  12. CNOT3 suppression promotes necroptosis by stabilizing mRNAs for cell death-inducing proteins

    PubMed Central

    Suzuki, Toru; Kikuguchi, Chisato; Sharma, Sahil; Sasaki, Toshio; Tokumasu, Miho; Adachi, Shungo; Natsume, Tohru; Kanegae, Yumi; Yamamoto, Tadashi

    2015-01-01

    The CCR4-NOT complex is conserved in eukaryotes and is involved in mRNA metabolism, though its molecular physiological roles remain to be established. We show here that CNOT3-depleted mouse embryonic fibroblasts (MEFs) undergo cell death. Levels of other complex subunits are decreased in CNOT3-depleted MEFs. The death phenotype is rescued by introduction of wild-type (WT), but not mutated CNOT3, and is not suppressed by the pan-caspase inhibitor, zVAD-fluoromethylketone. Gene expression profiling reveals that mRNAs encoding cell death-related proteins, including receptor-interacting protein kinase 1 (RIPK1) and RIPK3, are stabilized in CNOT3-depleted MEFs. Some of these mRNAs bind to CNOT3, and in the absence of CNOT3 their poly(A) tails are elongated. Inhibition of RIPK1-RIPK3 signaling by a short-hairpin RNA or a necroptosis inhibitor, necrostatin-1, confers viability upon CNOT3-depleted MEFs. Therefore, we conclude that CNOT3 targets specific mRNAs to prevent cells from being disposed to necroptotic death. PMID:26437789

  13. Polycistronic trypanosome mRNAs are a target for the exosome

    PubMed Central

    Kramer, Susanne; Piper, Sophie; Estevez, Antonio; Carrington, Mark

    2016-01-01

    Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNAs from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5′-3′ exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNAs. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. PMID:26946399

  14. Polycistronic trypanosome mRNAs are a target for the exosome.

    PubMed

    Kramer, Susanne; Piper, Sophie; Estevez, Antonio; Carrington, Mark

    2016-01-01

    Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNAs from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5'-3' exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNAs. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. PMID:26946399

  15. Mini-review: Making scent of the presence and local translation of odorant receptor mRNAs in olfactory axons

    PubMed Central

    Dubacq, Caroline; Fouquet, Coralie; Trembleau, Alain

    2016-01-01

    Rodents contain in their genome more than 1,000 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. PMID:23959692

  16. 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.

  17. Switches in gene expression including microRNA and a large number of distinct mRNAs

    NASA Astrophysics Data System (ADS)

    Zhdanov, V. P.

    2008-12-01

    In eukaryotic cells, the kinetics of gene expression depends on the interplay of messenger RNAs (mRNAs), proteins, and nonprotein coding RNAs, or, more specifically, microRNAs. Some microRNAs may target hundreds of mRNAs. To describe this case, the author proposes a kinetic model implying that the microRNA synthesis is suppressed by the protein produced via the translation of one of the target mRNAs. With physically reasonable model parameters, the model predicts bistability or, in other words, switches in the expression of hundreds of genes.

  18. Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat 1

    PubMed Central

    Helm, Kenneth W.; Abernethy, Rollin H.

    1990-01-01

    Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times. Images Figure 1 Figure 2 Figure 3

  19. ncRNA-mediated bistability in the synthesis of hundreds of distinct mRNAs and proteins

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2010-02-01

    The kinetics of gene expression can be bistable due to the feedback between the mRNA and protein formation. In eukaryotic cells, the interplay between mRNAs and proteins can be influenced by non-coding RNAs. Some of these RNAs, e.g., microRNAs, may target hundreds of distinct mRNAs. The model presented here shows how a non-coding RNA can be used as a mediator in order to involve numerous mRNAs and proteins into a bistable network.

  20. Characterization of binding of LARP6 to the 5’ stem-loop of collagen mRNAs: Implications for synthesis of type I collagen

    PubMed Central

    Stefanovic, Lela; Longo, Liam; Zhang, Yujie; Stefanovic, Branko

    2014-01-01

    Type I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their 5’ UTRs, the 5’SL. LARP6 binds the 5’SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5’SL contribute to the high affinity of LARP6 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. LARP6 binding to 5’SL of collagen α2(I) mRNA is more stable than the binding to 5’SL of α1(I) mRNA, although the equilibrium binding constants are similar. The more stable binding to α2(I) mRNA may favor synthesis of the heterotrimeric type I collagen. LARP6 needs 2 domains to contact 5’SL, the La domain and the RRM. T133 in the La domain is critical for folding of the protein, while loop 3 in the RRM is critical for binding 5’SL. Loop 3 is also involved in the interaction of LARP6 and protein translocation channel SEC61. This interaction is essential for type I collagen synthesis, because LARP6 mutant which binds 5’SL but which does not interact with SEC61, suppresses collagen synthesis in a dominant negative manner. We postulate that LARP6 directly targets collagen mRNAs to the SEC61 translocons to facilitate coordinated translation of the 2 collagen mRNAs. The unique sequences of LARP6 identified in this work may have evolved to enable its role in type I collagen biosynthesis. PMID:25692237

  1. 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

  2. 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. PMID:26369818

  3. 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

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

    PubMed

    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

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

    PubMed Central

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

    1995-01-01

    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. PMID:7549487

  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. PMID:26784711

  7. eIF2alpha kinases GCN2 and PERK modulate transcription and translation of distinct sets of mRNAs in mouse liver.

    PubMed

    Dang Do, An N; Kimball, Scot R; Cavener, Douglas R; Jefferson, Leonard S

    2009-08-01

    In eukaryotes, selective derepression of mRNA translation through altered utilization of upstream open reading frames (uORF) or internal ribosomal entry sites (IRES) regulatory motifs following exposure to stress is regulated at the initiation stage through the increased phosphorylation of eukaryotic initiation factor 2 on its alpha-subunit (eIF2alpha). While there is only one known eIF2alpha kinase in yeast, general control nonderepressible 2 (GCN2), mammals have evolved to express at least four: GCN2, heme-regulated inhibitor kinase (HRI), double-stranded RNA-activated protein kinase (PKR), and PKR-like endoplasmic reticulum-resident kinase (PERK). So far, the main known distinction among these four kinases is their activation in response to different acute stressors. In the present study, we used the in situ perfused mouse liver model and hybridization array analyses to assess the general translational response to stress regulated by two of these kinases, GCN2 and PERK, and to differentiate between the downstream effects of activating GCN2 versus PERK. The resulting data showed that at least 2.5% of mouse liver mRNAs are subject to derepressed translation following stress. In addition, the data demonstrated that eIF2alpha kinases GCN2 and PERK differentially regulate mRNA transcription and translation, which in the latter case suggests that increased eIF2alpha phosphorylation is not sufficient for derepression of translation. These findings open an avenue for more focused future research toward groups of mRNAs that code for the early cellular stress response proteins. PMID:19509078

  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. PMID:26950602

  10. Expression of type I and type V collagen mRNAs in the elasmoid scales of a teleost fish as revealed by in situ hybridization.

    PubMed

    Le Guellec, D; Zylberberg, L

    1998-01-01

    The ability of scale-forming cells to produce both type I and type V collagens was investigated by in situ hybridization at the light and electron microscope levels. Biochemical analyses reported that type I collagen, the predominant component, was associated with the minor type V collagen in the collagenous matrix of the teleost scales where, thin and thick collagen fibrils formed distinct layers. Thin collagen fibrils of the external layer were produced by the episquamal scleroblasts scattered on the outer scale surface, while thick collagen fibrils forming the compact basal plate were produced by the hyposquamal scleroblasts lining the inner surface of the scale. We demonstrated that episquamal and hyposquamal scleroblasts contained mRNAs for alpha1(I) and alpha1(V) collagens. Quantification by image analysis of the relative amount of alpha1(I) and alpha1(V) mRNAs in episquamal and hyposquamal scleroblasts suggests that the gene expression of type V collagen was proportionally higher in episquamal scleroblasts. These results support our hypothesis that the diameter of the thin fibrils of the external layer is regulated by the significant amount of type V collagen that interacts with type I collagen. PMID:11063006

  11. Electrophoretic separation of in vitro translation products on giant two-dimensional gels allows detailed analysis of cellular mRNAs

    SciTech Connect

    Colbert, R.A.; Young, D.A.

    1986-11-05

    The in vitro translation products of mRNA pretreated with methylmercuric hydroxide were examined by giant two-dimensional gel electrophoresis. In addition to increasing overall translational efficiency approximately 2.5-fold, methylmercuric hydroxide selectively increases the translation of mRNAs coding for higher molecular mass (greater than 45 kDa) proteins, allowing the routine resolution of 1500 (35S)methionine-labeled proteins. This yields 3 to 4-fold the number of translation products seen with smaller size two-dimensional gels. With this method we compare thymus cell proteins synthesized in vivo with the products of in vitro translation of mRNA recovered from thymus cells. Fifty-eight percent of the translation products are qualitatively the same as proteins synthesized in vivo (similar Mr, pI, and neighboring proteins), with 64% of these also being quantitatively similar (less than 5-fold difference). A comparison of thymus mRNA in vitro translation products with those coded for by mRNA from liver reveals only 32% qualitative similarity, with 63% of these also being quantitatively similar. These results are discussed in relation to predictions of mRNA abundance and complexity based on DNA:RNA hybridization data. Giant two-dimensional gel separations of in vitro translation products appear to be useful for detecting less abundant cellular mRNAs, including those that may be regulated by hormones or other physiological mediators.

  12. Aire-dependent peripheral tissue antigen mRNAs in mTEC cells feature networking refractoriness to microRNA interaction.

    PubMed

    Macedo, Claudia; Oliveira, Ernna H; Almeida, Renata S; Donate, Paula B; Fornari, Thaís A; Pezzi, Nicole; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Passos, Geraldo A

    2015-01-01

    The downregulation of PTA genes in mTECs is associated with the loss of self-tolerance, and the role of miRNAs in this process is not fully understood. Therefore, we studied the expression of mRNAs and miRNAs in mTECs from autoimmune NOD mice during the period when loss of self-tolerance occurs in parallel with non-autoimmune BALB/c mice. Although the expression of the transcriptional regulator Aire was unchanged, we observed downregulation of a set of PTA mRNAs. A set of miRNAs was also differentially expressed in these mice. The reconstruction of miRNA-mRNA interaction networks identified the controller miRNAs and predicted the PTA mRNA targets. Interestingly, the known Aire-dependent PTAs exhibited pronounced refractoriness in the networking interaction with miRNAs. This study reveals the existence of a new mechanism in mTECs, and this mechanism may have importance in the control of self-tolerance. PMID:25220732

  13. Splice junctions in adenovirus 2 early region 4 mRNAs: multiple splice sites produce 18 to 24 RNAs.

    PubMed Central

    Tigges, M A; Raskas, H J

    1984-01-01

    We localized the splice junctions in adenovirus 2 early region 4 (E4) mRNAs. Processing of the E4 precursor RNA positioned the donor splice site of the 5' leader sequence adjacent to acceptor sites near the 5' ends of five of the six open reading regions in the E4 transcription unit. Of particular interest among the E4 mRNAs is an extensively spliced class which includes multiple species with sizes ranging from 1.1 to 0.75 kilobases (kb). Purified 1.1- to 0.75-kb mRNAs specified at least 10 polypeptides in vitro. We detected eight acceptor and two donor splice sites utilized in the deletion of the intron from the 3' portion of these mRNAs. E4 RNAs were isolated from the cytoplasm of infected cells at 5, 9, 12, and 18 h after infection. The E4 mRNAs were present throughout infection, but different members of the 1.1- to 0.7-kb class were predominant at each time assayed. Alternate splicing of the 3.0-kb E4 precursor RNA can generate as many as 25 mRNAs that encode at least 16 polypeptides. Images PMID:6336328

  14. Expression of Procyclin mRNAs during Cyclical Transmission of Trypanosoma brucei

    PubMed Central

    2005-01-01

    Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3′ untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised. PMID:16276404

  15. Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

    PubMed Central

    Bouloy, Michele; Plotch, Stephen J.; Krug, Robert M.

    1978-01-01

    Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the α-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized in vitro primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5′-phosphate in the presence of S-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5′-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription in vitro. Images PMID:283399

  16. 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.

  17. 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.

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

    NASA Astrophysics Data System (ADS)

    Zhdanov, V. P.

    2010-10-01

    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.

  19. Determining exon connectivity in complex mRNAs by nanopore sequencing.

    PubMed

    Bolisetty, Mohan T; Rajadinakaran, Gopinath; Graveley, Brenton R

    2015-01-01

    Short-read high-throughput RNA sequencing, though powerful, is limited in its ability to directly measure exon connectivity in mRNAs that contain multiple alternative exons located farther apart than the maximum read length. Here, we use the Oxford Nanopore MinION sequencer to identify 7,899 'full-length' isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be a powerful method for comprehensive transcriptome characterization. PMID:26420219

  20. Profiles of nuclear and mitochondrial encoded mRNAs in developing and quiescent embryos of Artemia franciscana.

    PubMed

    Hardewig, I; Anchordoguy, T J; Crawford, D L; Hand, S C

    1996-05-24

    Embryos of the brine shrimp Artemia franciscana are able to withstand long bouts of environmental anoxia by entering a quiescent state during which metabolism is greatly depressed. Recent evidence supports a global arrest of protein synthesis during quiescence. In this study we measured the amounts of mRNA for a mitochondrial-encoded subunit of cytochrome c oxidase (COX I) and for nuclear-encoded actin during aerobic development, anaerobiosis, and aerobic acidosis (artificial quiescence imposed by intracellular acidification under aerobic conditions). The levels of both COX I and actin transcripts increased significantly during aerobic development. COX I mRNA levels were tightly correlated with previous measures of COX catalytic activity, which suggests that COX synthesis could be regulated by message concentration during aerobic development. The ontogenetic increase for these mRNAs was blocked by anoxia and aerobic acidosis. Importantly, the levels of COX I and actin mRNA did not decline appreciably during the 6 h bouts of quiescence, even though protein synthesis is acutely arrested by these same treatments. Thus, the constancy of mRNA levels during quiescence indicate that reduced protein synthesis is not caused by message limitation, but rather, is likely controlled at the translational level. One advantage of this regulatory mechanism is the conservation of mRNA molecules during quiescence, which would potentially favor a quick resumption of translation as soon as oxygen is returned to the embryos. Finally, because anoxia and aerobic acidosis are both characterized by acidic intracellular pH, the reduction in pH may serve, directly or indirectly, as one signal regulating levels of mRNA in this embryo during quiescence. PMID:8817476

  1. Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs

    PubMed Central

    Durand, Sylvain; Richard, Graziella; Bontems, François; Uzan, Marc

    2012-01-01

    The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine–Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5′-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5′-monophosphate termini, to attack mRNAs from the 5′-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5′ terminus for mRNA stability and depicts a pathway of mRNA degradation with 5′- to 3′-polarity in cells devoid of 5′–3′ exonucleases. It also ascribes a role for T4 PNK during normal phage development. PMID:22499790

  2. Single molecule tracking of quantum dot-labeled mRNAs in a cell nucleus

    SciTech Connect

    Ishihama, Yo; Funatsu, Takashi

    2009-03-27

    Single particle tracking (SPT) is a powerful technique for studying mRNA dynamics in cells. Although SPT of mRNA has been performed by labeling mRNA with fluorescent dyes or proteins, observation of mRNA for long durations with high temporal resolution has been difficult due to weak fluorescence and rapid photobleaching. Using quantum dots (QDs), we succeeded in observing the movement of individual mRNAs for more than 60 s, with a temporal resolution of 30 ms. Intronless and truncated ftz mRNA, synthesized in vitro and labeled with QDs, was microinjected into the nuclei of Cos7 cells. Almost all mRNAs were in motion, and statistical analyses revealed anomalous diffusion between barriers, with a microscopic diffusion coefficient of 0.12 {mu}m{sup 2}/s and a macroscopic diffusion coefficient of 0.025 {mu}m{sup 2}/s. Diffusion of mRNA was observed in interchromatin regions but not in histone2B-GFP-labeled chromatin regions. These results provide direct evidence of channeled mRNA diffusion in interchromatin regions.

  3. Genome-Wide Analysis Reveals Selective Modulation of microRNAs and mRNAs by Histone Deacetylase Inhibitor in B Cells Induced to Undergo Class-Switch DNA Recombination and Plasma Cell Differentiation

    PubMed Central

    Shen, Tian; Sanchez, Helia N.; Zan, Hong; Casali, Paolo

    2015-01-01

    As we have suggested, epigenetic factors, such as microRNAs (miRNAs), can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase (HDAC) inhibitors (HDI) inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR), somatic hypermutation (SHM), and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J Immunol 193:5933–5950, 2014). To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, and increased expression of miR-155, miR-181b, and miR-361, which target Aicda, and miR-23b, miR-30a, and miR-125b, which target Prdm1. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell). Only 18 (0.36%) of these highly expressed mRNAs, including Aicda, Prdm1, and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30%) of the highly expressed mRNAs were upregulated (more than twofold) by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting, or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or

  4. Genome-Wide Analysis Reveals Selective Modulation of microRNAs and mRNAs by Histone Deacetylase Inhibitor in B Cells Induced to Undergo Class-Switch DNA Recombination and Plasma Cell Differentiation.

    PubMed

    Shen, Tian; Sanchez, Helia N; Zan, Hong; Casali, Paolo

    2015-01-01

    As we have suggested, epigenetic factors, such as microRNAs (miRNAs), can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase (HDAC) inhibitors (HDI) inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR), somatic hypermutation (SHM), and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J Immunol 193:5933-5950, 2014). To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, and increased expression of miR-155, miR-181b, and miR-361, which target Aicda, and miR-23b, miR-30a, and miR-125b, which target Prdm1. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell). Only 18 (0.36%) of these highly expressed mRNAs, including Aicda, Prdm1, and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30%) of the highly expressed mRNAs were upregulated (more than twofold) by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting, or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or

  5. Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.

    PubMed Central

    Sellami, M; Rether, B; Gangloff, J; Ebel, J P; Bonnet, J

    1983-01-01

    We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs. Images PMID:6344009

  6. Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli

    PubMed Central

    Vesper, Oliver; Amitai, Shahar; Belitsky, Maria; Byrgazov, Konstantin; Kaberdina, Anna Chao; Engelberg-Kulka, Hanna; Moll, Isabella

    2016-01-01

    Summary Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3′ terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli. PMID:21944167

  7. Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

    PubMed Central

    Alexandersen, S; Carpenter, S; Christensen, J; Storgaard, T; Viuff, B; Wannemuehler, Y; Belousov, J; Roth, J A

    1993-01-01

    The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown. Images PMID:8380084

  8. 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. PMID:25086243

  9. Sequence analysis and compositional properties of untranslated regions of human mRNAs.

    PubMed

    Pesole, G; Fiormarino, G; Saccone, C

    1994-03-25

    A detailed computer analysis of the untranslated regions, 5'-UTR and 3'-UTR, of human mRNA sequences is reported. The compositional properties of these regions, compared with those of the corresponding coding regions, indicate that 5'-UTR and 3'-UTR are less affected by the isochore compartmentalization than the corresponding third codon positions of mRNAs. The presence of higher functional constraints in 5'-UTR is also reported. Dinucleotide analysis shows a depletion of CpG and TpA in both sequences. A search for significant sequence motifs using the WORDUP algorithm reveals the patterns already known to have a functional role in the mRNA UTR, and several other motifs whose functional roles remain to be demonstrated. This type of analysis may be particularly useful for guiding site-directed mutagenesis experiments. In addition, it can be used for assessing the nature of anonymous sequences now produced in large amounts in megabase sequencing projects. PMID:8144029

  10. Targeting cellular mRNAs translation by CRISPR-Cas9

    PubMed Central

    Liu, Yuchen; Chen, Zhicong; He, Anbang; Zhan, Yonghao; Li, Jianfa; Liu, Li; Wu, Hanwei; Zhuang, Chengle; Lin, Junhao; Zhang, Qiaoxia; Huang, Weiren

    2016-01-01

    Recently CRISPR-Cas9 system has been reported to be capable of targeting a viral RNA, and this phenomenon thus raises an interesting question of whether Cas9 can also influence translation of cellular mRNAs. Here, we show that both natural and catalytically dead Cas9 can repress mRNA translation of cellular genes, and that only the first 14 nt in the 5′ end of sgRNA is essential for this process. CRISPR-Cas9 can suppress the protein expression of an unintended target gene without affecting its DNA sequence and causes unexpected phenotypic changes. Using the designed RNA aptamer-ligand complexes which physically obstruct translation machinery, we indicate that roadblock mechanism is responsible for this phenomenon. Our work suggests that studies on Cas9 should avoid the potential off-target effects by detecting the alteration of genes at both the DNA and protein levels. PMID:27405721

  11. Preferential Translation of Vesicular Stomatitis Virus mRNAs Is Conferred by Transcription from the Viral Genome▿

    PubMed Central

    Whitlow, Zackary W.; Connor, John H.; Lyles, Douglas S.

    2006-01-01

    Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs. PMID:17005665

  12. Direct Reprogramming of Human Fibroblasts to Hepatocyte-Like Cells by Synthetic Modified mRNAs

    PubMed Central

    Simeonov, Kamen P.; Uppal, Hirdesh

    2014-01-01

    Direct reprogramming by overexpression of defined transcription factors is a promising new method of deriving useful but rare cell types from readily available ones. While the method presents numerous advantages over induced pluripotent stem (iPS) cell approaches, a focus on murine conversions and a reliance on retroviral vectors limit potential human applications. Here we address these concerns by demonstrating direct conversion of human fibroblasts to hepatocyte-like cells via repeated transfection with synthetic modified mRNAs. Hepatic induction was achieved with as little as three transcription factor mRNAs encoding HNF1A plus any two of the factors, FOXA1, FOXA3, or HNF4A in the presence of an optimized hepatic growth medium. We show that the absolute necessity of exogenous HNF1A mRNA delivery is explained both by the factor's inability to be activated by any other factors screened and its simultaneous ability to strongly induce expression of other master hepatic transcription factors. Further analysis of factor interaction showed that a series of robust cross-activations exist between factors that induce a hepatocyte-like state. Transcriptome and small RNA sequencing during conversion toward hepatocyte-like cells revealed global preferential activation of liver genes and miRNAs over those associated with other endodermal tissues, as well as downregulation of fibroblast-associated genes. Induced hepatocyte-like cells also exhibited hepatic morphology and protein expression. Our data provide insight into the process by which direct hepatic reprogramming occurs in human cells. More importantly, by demonstrating that it is possible to achieve direct reprogramming without the use of retroviral gene delivery, our results supply a crucial step toward realizing the potential of direct reprogramming in regenerative medicine. PMID:24963715

  13. Annexin II is associated with mRNAs which may constitute a distinct subpopulation.

    PubMed Central

    Vedeler, A; Hollås, H

    2000-01-01

    Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs. PMID:10839987

  14. 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. PMID:25858778

  15. Deep sequencing shows multiple oligouridylations are required for 3' to 5' degradation of histone mRNAs on polyribosomes.

    PubMed

    Slevin, Michael K; Meaux, Stacie; Welch, Joshua D; Bigler, Rebecca; Miliani de Marval, Paula L; Su, Wei; Rhoads, Robert E; Prins, Jan F; Marzluff, William F

    2014-03-20

    Histone mRNAs are rapidly degraded when DNA replication is inhibited during S phase with degradation initiating with oligouridylation of the stem loop at the 3' end. We developed a customized RNA sequencing strategy to identify the 3' termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3' side of the stem loop that resulted from initial degradation by 3'hExo and intermediates near the stop codon and within the coding region. Sequencing of polyribosomal histone mRNAs revealed that degradation initiates and proceeds 3' to 5' on translating mRNA and that many intermediates are capped. Knockdown of the exosome-associated exonuclease PM/Scl-100, but not the Dis3L2 exonuclease, slows histone mRNA degradation consistent with 3' to 5' degradation by the exosome containing PM/Scl-100. Knockdown of No-go decay factors also slowed histone mRNA degradation, suggesting a role in removing ribosomes from partially degraded mRNAs. PMID:24656133

  16. 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.

  17. Translation efficiency of adenovirus early region 1A mRNAs deleted in the 5' untranslated region.

    PubMed Central

    Spindler, K R; Berk, A J

    1984-01-01

    Adenovirus deletion mutants were studied to examine the influence of the 5' untranslated sequence on the translation of early region 1A mRNAs. Alterations of the 5' untranslated sequence, including complete deletion of the wild-type 5' untranslated sequence, did not significantly affect the rate of translation. Images PMID:6471170

  18. 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...

  19. Organization of early region 1B of human adenovirus type 2: identification of four differentially spliced mRNAs.

    PubMed Central

    Virtanen, A; Pettersson, U

    1985-01-01

    The mRNAs from early region 1B of adenovirus type 2 have been studied by Northern blot, S1 nuclease, and cDNA analysis. Two novel mRNAs, designated 14S and 14.5S, have been observed in addition to the previously identified 9S, 13S, and 22S mRNAs. They are 1.26 and 1.31 kilobases long and differ from the 13S and 22S mRNAs in being composed of three exons instead of two. Their two terminal exons are the same as those present in the 13S mRNA, whereas the middle exon is unique to each of the two novel mRNA species. The structures of the 14S and 14.5S mRNAs allow the prediction of their coding capacities: both mRNA species, like the 22S and 13S mRNAs, contain an uninterrupted translational reading frame encoding a 21,000-molecular-weight (21K) polypeptide. The 14S mRNA can, in addition, encode a 16.5K polypeptide which shares N-terminal and C-terminal sequences with the 55K polypeptide, known to be encoded by the 22S mRNA. The 14.5S mRNA species encodes a hypothetical 9.2K polypeptide which has the same N terminus as the 55K polypeptide but a unique C terminus. The two mRNAs differ in their kinetics of appearance; the 14.5S mRNA is preferentially expressed late after infection in contrast to the 14S mRNA, which is present in approximately equal amounts early and late after infection. Taken together with previously published information the results suggest that early region 1B of adenovirus type 2 encodes five proteins in addition to virion polypeptide IX. These have predicted molecular weights of 55,000, 21,000, 16,500, 9,200, and 8,100. Images PMID:3989911

  20. Mature maternal mRNAs are longer than zygotic ones and have complex degradation kinetics in sea urchin.

    PubMed

    Gildor, Tsvia; Malik, Assaf; Sher, Noa; Ben-Tabou de-Leon, Smadar

    2016-06-01

    Early in embryogenesis, maternally deposited transcripts are degraded and new zygotic transcripts are generated during the maternal to zygotic transition. Recent works have shown that early zygotic transcripts are short compared to maternal transcripts, in zebrafish and Drosophila species. The reduced zygotic transcript length was attributed to the short cell cycle in these organisms that prevents the transcription of long primary transcripts (intron delay). Here we study the length of maternal mRNAs and their degradation kinetics in two sea urchin species to further the understanding of maternal gene usage and processing. Early zygotic primary transcripts and mRNAs are shorter than maternal ones in the sea urchin, Strongylocentrotus purpuratus. Yet, while primary transcripts length increases when cell cycle lengthens, typical for intron delay, the relatively short length of zygotic mRNAs is consistent. The enhanced mRNA length is due to significantly longer maternal open reading frames and 3'UTRs compared to the zygotic lengths, a ratio that does not change with developmental time. This implies unique usage of both coding sequences and regulatory information in the maternal stage compared to the zygotic stages. We extracted the half-lifetimes due to maternal and zygotic degradation mechanisms from high-density time course of a set of maternal mRNAs in Paracentrotus lividus. The degradation rates due to maternal and zygotic degradation mechanisms are not correlated, indicating that these mechanisms are independent and relay on different regulatory information. Our studies illuminate specific structural and kinetic properties of sea urchin maternal mRNAs that might be broadly shared by other organisms. PMID:27085752

  1. 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. PMID:26148972

  2. The BTG4 and CAF1 complex prevents the spontaneous activation of eggs by deadenylating maternal mRNAs.

    PubMed

    Pasternak, Michał; Pfender, Sybille; Santhanam, Balaji; Schuh, Melina

    2016-09-01

    Once every menstrual cycle, eggs are ovulated into the oviduct where they await fertilization. The ovulated eggs are arrested in metaphase of the second meiotic division, and only complete meiosis upon fertilization. It is crucial that the maintenance of metaphase arrest is tightly controlled, because the spontaneous activation of the egg would preclude the development of a viable embryo (Zhang et al. 2015 J. Genet. Genomics 42, 477-485. (doi:10.1016/j.jgg.2015.07.004); Combelles et al. 2011 Hum. Reprod. 26, 545-552. (doi:10.1093/humrep/deq363); Escrich et al. 2011 J. Assist. Reprod. Genet. 28, 111-117. (doi:10.1007/s10815-010-9493-5)). However, the mechanisms that control the meiotic arrest in mammalian eggs are only poorly understood. Here, we report that a complex of BTG4 and CAF1 safeguards metaphase II arrest in mammalian eggs by deadenylating maternal mRNAs. As a follow-up of our recent high content RNAi screen for meiotic genes (Pfender et al. 2015 Nature 524, 239-242. (doi:10.1038/nature14568)), we identified Btg4 as an essential regulator of metaphase II arrest. Btg4-depleted eggs progress into anaphase II spontaneously before fertilization. BTG4 prevents the progression into anaphase by ensuring that the anaphase-promoting complex/cyclosome (APC/C) is completely inhibited during the arrest. The inhibition of the APC/C relies on EMI2 (Tang et al. 2010 Mol. Biol. Cell 21, 2589-2597. (doi:10.1091/mbc.E09-08-0708); Ohe et al. 2010 Mol. Biol. Cell 21, 905-913. (doi:10.1091/mbc.E09-11-0974)), whose expression is perturbed in the absence of BTG4. BTG4 controls protein expression during metaphase II arrest by forming a complex with the CAF1 deadenylase and we hypothesize that this complex is recruited to the mRNA via interactions between BTG4 and poly(A)-binding proteins. The BTG4-CAF1 complex drives the shortening of the poly(A) tails of a large number of transcripts at the MI-MII transition, and this wave of deadenylation is essential for the arrest in

  3. 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.

  4. 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

  5. Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A.

    PubMed Central

    Krause, J E; Chirgwin, J M; Carter, M S; Xu, Z S; Hershey, A D

    1987-01-01

    Synthetic oligonucleotides were used to screen a rat striatal cDNA library for sequences corresponding to the tachykinin peptides substance P and neurokinin A. The cDNA library was constructed from RNA isolated from the rostral portion of the rat corpus striatum, the site of striatonigral cell bodies. Two types of cDNAs were isolated and defined by restriction enzyme analysis and DNA sequencing to encode both substance P and neurokinin A. The two predicted preprotachykinin protein precursors (130 and 115 amino acids in length) differ from each other by a pentadecapeptide sequence between the two tachykinin sequences, and both precursors possess appropriate processing signals for substance P and neurokinin A production. The presence of a third preprotachykinin mRNA of minor abundance in rat striatum was established by S1 nuclease protection experiments. This mRNA encodes a preprotachykinin of 112 amino acids containing substance P but not neurokinin A. These three mRNAs are derived from one rat gene as a result of differential RNA processing; thus, this RNA processing pattern further increases the diversity of products that can be generated from the preprotachykinin gene. Images PMID:2433692

  6. Complete set of mitochondrial pan-edited mRNAs in Leishmania mexicana amazonensis LV78

    PubMed Central

    Maslov, Dmitri A.

    2010-01-01

    Editing of mRNA transcribed from the mitochondrial cryptogenes ND8 (G1), ND9 (G2), G3, G4, ND3 (G5), RPS12 (G6) was investigated in Leishmania mexicana amazonensis, strain LV78, by amplification of the cDNA, cloning and sequencing. For each of these genes, extensively and partially edited transcripts were found to be relatively abundant compared to the respective pre-edited molecules. Moreover, the editing patterns observed in a majority of transcripts of each gene were consistent among themselves which allowed for inferring consensus editing sequences. The open reading frames contained in the consensus sequences were predicted to encode polypeptides that were highly similar to their counterparts in other species of Trypanosomatidae. Several kinetoplast DNA minicircles from this species available in the public domain were found to contain genes for guide RNAs which mediate editing of some of the mRNAs. The results indicate that the investigated strain of L. m. amazonensis has preserved its full editing capacity in spite of the long-term maintenance in culture. This property differs drastically from the other Leishmania species which lost some or all of the G1–G5 mRNA editing ability in culture. PMID:20546801

  7. Polymorphic CUG repeats in human mRNAs and their effects on gene expression.

    PubMed

    Tian, Bin; Mukhopadhyay, Rupa; Mathews, Michael B

    2005-01-01

    Expanded CUG repeats in the 3'-untranslated region (UTR) of the gene encoding myotonic dystrophy protein kinase (DMPK) cause myotonic dystrophy type 1 disease (DM1). The presence of such repeats has been found to impede gene expression at several levels in model systems. We took a bioinformatic approach to survey all human mRNA sequences for polymorphic CUG repeats. Our survey revealed that CUG repeats occur widely in various regions of mRNAs, with higher frequency in protein coding regions than 5'-UTRs or 3'-UTRs. About 30 genes were found to contain CUG repeats that are polymorphic in the number of repeats, suggesting the potential to expand or shrink. However, long polymorphic repeats were restricted to the 3'-UTR of the DMPK gene and the coding region of the ribosomal protein L14 gene. Using cell-free translation systems, we showed that extended CUG repeats can inhibit protein synthesis in vitro in the rabbit reticulocyte lysate, but not in wheat germ extracts, consistent with our previous finding of an interaction of CUG repeats with the protein kinase PKR. In transfected cells, CUG repeats can inhibit gene expression both in cis and in trans. However, observations with PKR-minus cells indicate that these effects are not primarily attributable to the interaction of extended CUG repeats with PKR. Northwestern blotting detected the presence in human cells of more CUG-binding proteins than are currently known. PMID:17114933

  8. Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection

    PubMed Central

    Showalter, Allan M.; Bell, John N.; Cramer, Carole L.; Bailey, John A.; Varner, Joseph E.; Lamb, Chris J.

    1985-01-01

    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 32P-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. Images PMID:16593612

  9. 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

  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. PMID:26528758

  11. Human proviral mRNAs down regulated in choriocarcinoma encode a zinc finger protein related to Krüppel.

    PubMed Central

    Kato, N; Shimotohno, K; VanLeeuwen, D; Cohen, M

    1990-01-01

    RNA transcripts of the HERV-R (ERV3) human provirus that are abundant in placenta but absent in choriocarcinoma contain nonproviral genomic sequences at their 3' ends. We report here the isolation of cDNA clones of these genomic sequences. The transcripts encode a Krüppel-related zinc finger protein consisting of a unique leader region and more than 12 28-amino-acid finger motifs. Images PMID:2115127

  12. Competition between splicing and polyadenylation reactions determines which adenovirus region E3 mRNAs are synthesized

    SciTech Connect

    Brady, H.A.; Wold, W.S.M.

    1988-08-01

    Complex transcription units encode multiple mRNAs which arise by alternative processing of a common pre-mRNA precursor. It is not known how the pre-mRNA processing pathways are determined or controlled. The authors are investigating this problem by using the E3 complex transcription unit of adenovirus as a model. Their approach is to construct virus mutants with lesions in E3 and then determine how the mutation affects the accumulation of E3 mRNAs in vivo. They report results which indicate that competition between splicing reactions and polyadenylation reactions occurs in vivo and that this plays an important role in alternative pre-mRNA processing.

  13. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.

    PubMed

    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

  14. Simultaneous Visualization of Multiple mRNAs and Matrix Metalloproteinases in Living Cells Using a Fluorescence Nanoprobe.

    PubMed

    Pan, Wei; Yang, Huijun; Li, Na; Yang, Limin; Tang, Bo

    2015-04-13

    Simultaneous monitoring of multiple tumour markers is of great significance for improving the accuracy of early cancer detection. In this study, a fluorescence nanoprobe has been prepared that can simultaneously monitor and visualize multiple mRNAs and matrix metalloproteinases (MMPs) in living cells. Confocal fluorescence imaging results indicate that the nanoprobe could effectively distinguish between cancer cells and normal cells even if one tumour maker of normal cells was overexpressed. Furthermore, it can detect changes in the expression levels of mRNAs and MMPs in living cells. The current approach could provide new tools for early cancer detection and monitoring the changes in expression levels of biomarkers during tumour progression. PMID:25752514

  15. On the nature and differential distribution of mRNAs in hippocampal neurites: implications for neuronal functioning.

    PubMed Central

    Miyashiro, K; Dichter, M; Eberwine, J

    1994-01-01

    Neurons are highly polarized cells with a mosaic of cytoplasmic and membrane proteins differentially distributed in axons, dendrites, and somata. In Drosophila and Xenopus, mRNA localization coupled with local translation is a powerful mechanism by which regionalized domains of surface or cytoplasmic proteins are generated. In neurons, there is substantial ultrastructural evidence positing the presence of protein synthetic machinery in neuronal processes, especially at or near postsynaptic sites. There are, however, remarkably few reports of mRNAs localized to these regions. We now present direct evidence that an unexpectedly large number of mRNAs, including members of the glutamate receptor family, second messenger system, and components of the translational control apparatus, are present in individual processes of hippocampal cells in culture. Images PMID:7971965

  16. Rhn1, a Nuclear Protein, Is Required for Suppression of Meiotic mRNAs in Mitotically Dividing Fission Yeast

    PubMed Central

    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. PMID:22912768

  17. RNA Sequencing and Proteogenomics Reveal the Importance of Leaderless mRNAs in the Radiation-Tolerant Bacterium Deinococcus deserti

    PubMed Central

    de Groot, Arjan; Roche, David; Fernandez, Bernard; Ludanyi, Monika; Cruveiller, Stéphane; Pignol, David; Vallenet, David; Armengaud, Jean; Blanchard, Laurence

    2014-01-01

    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. PMID:24723731

  18. 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. PMID:23519614

  19. Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific

    PubMed Central

    Wang, Tong; Cui, Yizhi; Jin, Jingjie; Guo, Jiahui; Wang, Guibin; Yin, Xingfeng; He, Qing-Yu; Zhang, Gong

    2013-01-01

    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 R2 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. PMID:23519614

  20. 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.

  1. Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruption.

    PubMed

    Davies, Ben; Behnen, Martina; Cappallo-Obermann, Heike; Spiess, Andrej-Nikolai; Theuring, Franz; Kirchhoff, Christiane

    2007-05-01

    Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the beta-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates. PMID:17034053

  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. PMID:26373828

  3. Most mammalian mRNAs are conserved targets of microRNAs

    PubMed Central

    Friedman, Robin C.; Farh, Kyle Kai-How; Burge, Christopher B.; Bartel, David P.

    2009-01-01

    MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2–7), particularly those in 3′ untranslated regions (3′UTRs), are preferentially conserved. Here, we overhauled our tool for finding preferential conservation of sequence motifs and applied it to the analysis of human 3′UTRs, increasing by nearly threefold the detected number of preferentially conserved miRNA target sites. The new tool more efficiently incorporates new genomes and more completely controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. The improved background model enabled preferential conservation of a new site type, the “offset 6mer,” to be detected. In total, >45,000 miRNA target sites within human 3′UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. Mammalian-specific miRNAs have far fewer conserved targets than do the more broadly conserved miRNAs, even when considering only more recently emerged targets. Although pairing to the 3′ end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites detected. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (PCT) correlating with experimental measurements of repression. Our expanded set of target predictions (including conserved 3′-compensatory sites), are available at the TargetScan website, which displays the PCT for each site and each predicted target. PMID:18955434

  4. 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

  5. Rotavirus Prevents the Expression of Host Responses by Blocking the Nucleocytoplasmic Transport of Polyadenylated mRNAs

    PubMed Central

    Rubio, Rosa M.; Mora, Silvia I.; Romero, Pedro; Arias, Carlos F.

    2013-01-01

    Rotaviruses are the most important agent of severe gastroenteritis in young children. Early in infection, these viruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis while viral proteins are efficiently synthesized. In infected cells, there is an accumulation of the cytoplasmic poly(A)-binding protein in the nucleus, induced by the viral protein NSP3. Here we found that poly(A)-containing mRNAs also accumulate and become hyperadenylated in the nuclei of infected cells. Using reporter genes bearing the untranslated regions (UTRs) of cellular or viral genes, we found that the viral UTRs do not determine the efficiency of translation of mRNAs in rotavirus-infected cells. Furthermore, we showed that while a polyadenylated reporter mRNA directly delivered into the cytoplasm of infected cells was efficiently translated, the same reporter introduced as a plasmid that needs to be transcribed and exported to the cytoplasm was poorly translated. Altogether, these results suggest that nuclear retention of poly(A)-containing mRNAs is one of the main strategies of rotavirus to control cell translation and therefore the host antiviral and stress responses. PMID:23536677

  6. Two Chlamydomonas OPR proteins stabilize chloroplast mRNAs encoding small subunits of photosystem II and cytochrome b6 f.

    PubMed

    Wang, Fei; Johnson, Xenie; Cavaiuolo, Marina; Bohne, Alexandra-Viola; Nickelsen, Joerg; Vallon, Olivier

    2015-06-01

    In plants and algae, chloroplast gene expression is controlled by nucleus-encoded proteins that bind to mRNAs in a specific manner, stabilizing mRNAs or promoting their splicing, editing, or translation. Here, we present the characterization of two mRNA stabilization factors of the green alga Chlamydomonas reinhardtii, which both belong to the OctotricoPeptide Repeat (OPR) family. MCG1 is necessary to stabilize the petG mRNA, encoding a small subunit of the cytochrome b6 f complex, while MBI1 stabilizes the psbI mRNA, coding for a small subunit of photosystem II. In the mcg1 mutant, the small RNA footprint corresponding to the 5'-end of the petG transcript is reduced in abundance. In both cases, the absence of the small subunit perturbs assembly of the cognate complex. Whereas PetG is essential for formation of a functional cytochrome b6 f dimer, PsbI appears partly dispensable as a low level of PSII activity can still be measured in its absence. Thus, nuclear control of chloroplast gene expression is not only exerted on the major core subunits of the complexes, but also on small subunits with a single transmembrane helix. While OPR proteins have thus far been involved in translation or trans-splicing of plastid mRNAs, our results expand the potential roles of this repeat family to their stabilization. PMID:25898982

  7. Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display.

    PubMed

    Wilkinson, J Q; Lanahan, M B; Conner, T W; Klee, H J

    1995-03-01

    Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. In climateric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. To gain insight into the molecular determinants associated with nonclimacteric fruit ripening, we examined mRNA populations in ripening strawberry fruit using polymerase chain reaction (PCR) differential display. Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appear to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool to study fruit ripening and other developmental processes in plants at the RNA level. PMID:7766892

  8. 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

  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. Nuclear accumulation of mRNAs underlies G4C2-repeat-induced translational repression in a cellular model of C9orf72 ALS.

    PubMed

    Rossi, Simona; Serrano, Alessia; Gerbino, Valeria; Giorgi, Alessandra; Di Francesco, Laura; Nencini, Monica; Bozzo, Francesca; Schininà, Maria Eugenia; Bagni, Claudia; Cestra, Gianluca; Carrì, Maria Teresa; Achsel, Tilmann; Cozzolino, Mauro

    2015-05-01

    A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS. PMID:25788698

  11. Occurrence of androgen and estrogen receptor mRNAs in the harderian gland: a comparative survey.

    PubMed

    Varriale, B

    1996-06-01

    In Rana esculenta the presence of an androgen receptor in both the male and female Harderian gland (HG) has been demonstrated. Hybridization analysis has evidenced a high degree of homology between the rat androgen receptor cDNA and the frog androgen receptor mRNA (fARmRNA). Correspondingly the molecular size of fARmRNA is similar to those described in mammals (9.4 kb). In in vivo experiments testosterone (T) increases the levels of fARmRNA. The use of the antiandrogen alone or in combination with T prevents the increase of fARmRNA. In the control animals a loss of fARmRNA has been observed. In primary cultures of HG cells, the steady-state levels of fARmRNA increase in the cells exposed to T. These results suggest that T exerts an autoinduction on its own receptor, increasing the levels of fARmRNA. In Xenopus laevis the HG shows a sexual dimorphism of the protein pattern. The female shows two major proteins (210 and 180 kDa). Administration of estradiol to the male shifts the protein pattern into the female one. In this respect an estrogen receptor mRNA (ERmRNA) has been found in the female gland and can be induced in the male one. No ARmRNA has been detected in either sexes. A similar sex dimorphism has been found in Gallus domesticus. The female pattern is characterised by a protein fraction of about 210 kDa, the male one by a protein fraction of about 180 kDa. In 4-day-old chicks no sex differences have been found. An ERmRNA is expressed in the female, while no ARmRNA has been detected in both sexes. Neither AR nor ER mRNAs have been detected in the chick HG. Among mammals the HG or the hamster (Mesocricetus auratus) shows an androgen-dependent sex dimorphism. In in vitro experiments T 10(-12) M induces a onefold increase of ARm-RNA with respect to unexposed cells. This effect reaches its maximum (4.4-fold) when cells are exposed to T 10(-8) M. The size of the hamster ARmRNA is similar to that observed in other mammals (9.5 kb). The above results suggest that

  12. Cross-talks between microRNAs and mRNAs in pancreatic tissues of streptozotocin-induced type 1 diabetic mice

    PubMed Central

    TIAN, CAIMING; OUYANG, XIAOXI; LV, QING; ZHANG, YAOU; XIE, WEIDONG

    2015-01-01

    Network cross-talks between microRNAs (miRNAs) and mRNAs may be useful to elucidate the pathological mechanisms of pancreatic islet cells in diabetic individuals. The aim of the present study was to investigate the cross-talks between miRNAs and mRNAs in pancreatic tissues of streptozotocin-induced diabetic mice through microarray and bioinformatic methods. Based on the miRNA microarray, 64 upregulated and 72 downregulated miRNAs were observed in pancreatic tissues in diabetic mice compared to the normal controls. Based on the mRNA microarrray, 507 upregulated mRNAs and 570 downregulated mRNAs were identified in pancreatic tissues in diabetic mice compared to the normal controls. Notably, there were 246 binding points between upregulated miRNA and downregulated mRNAs; simultaneously, there were 583 binding points between downregulated miRNA and upregulated mRNAs. These changed mRNA may potentially involve the following signaling pathways: Insulin secretion, pancreatic secretion, mammalian target of rapamycin signaling pathway, forkhead box O signaling pathway and phosphatidylinositol 3-kinase-protein kinase B signaling. The fluctuating effects of miRNAs and matched mRNAs indicated that miRNAs may have wide cross-talks with mRNAs in pancreatic tissues of type 1 diabetic mice. The cross-talks may play important roles in contributing to impaired islet functions and the development of diabetes. However, further functional validation should be conducted in the future. PMID:26137232

  13. The RNA Binding Protein Tudor-SN Is Essential for Stress Tolerance and Stabilizes Levels of Stress-Responsive mRNAs Encoding Secreted Proteins in Arabidopsis[C][W][OA

    PubMed Central

    dit Frey, Nicolas Frei; Muller, Philippe; Jammes, Fabien; Kizis, Dimosthenis; Leung, Jeffrey; Perrot-Rechenmann, Catherine; Bianchi, Michele Wolfe

    2010-01-01

    Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death–associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway. PMID:20484005

  14. Increased food intake stimulates GnRH-I, glycoprotein hormone alpha-subunit and follistatin mRNAs, and ovarian follicular numbers in laying broiler breeder hens.

    PubMed

    Ciccone, N A; Dunn, I C; Sharp, P J

    2007-07-01

    The aim of this study, in 36 week-old laying broiler breeder hens, was to establish the effects on reproductive neuroendocrine gene expression of reinstating ad libitum food intake after moderate food restriction from 2 weeks of age. Seven days of ad libitum feeding increased the number of large pre-ovulatory ovarian follicles and gonadotropin releasing hormone-I (GnRH-I), glycoprotein hormone alpha-subunit and follistatin mRNAs. Plasma luteinizing hormone (LH) was also increased while plasma follicle-stimulating hormone (FSH) was reduced. There were no associated changes in gonadotropin inhibitory hormone (GnIH), LHbeta or FSHbeta mRNAs. The mechanism underlying the increased expression of alpha-subunit and follistatin mRNAs was investigated in vitro by incubating pituitary fragments with pulses of GnRH-I. This treatment increased alpha-subunit and follistatin mRNAs but did not affect gonadotropin beta-subunit mRNAs. It is concluded that lifting food restriction in laying hens increases GnRH-I gene transcription or mRNA stability which may be a consequence, or cause of increased GnRH-I release. This, in turn, increases glycoprotein hormone alpha-subunit and follistatin mRNAs, resulting in increased plasma LH and decreased plasma FSH, respectively. PMID:16737793

  15. Expression Profiling of Differentiating Eosinophils in Bone Marrow Cultures Predicts Functional Links between MicroRNAs and Their Target mRNAs

    PubMed Central

    Eyers, Fiona; Xiang, Yang; Guo, Man; Young, Ian G.; Rosenberg, Helene F.

    2014-01-01

    Background MicroRNAs (miRNAs) are small non-coding RNAs that regulate complex transcriptional networks underpin immune responses. However, little is known about the specific miRNA networks that control differentiation of specific leukocyte subsets. In this study, we profiled miRNA expression during differentiation of eosinophils from bone marrow (BM) progenitors (bmEos), and correlated expression with potential mRNA targets involved in crucial regulatory functions. Profiling was performed on whole BM cultures to document the dynamic changes in miRNA expression in the BM microenvironment over the differentiation period. miRNA for network analysis were identified in BM cultures enriched in differentiating eosinophils, and chosen for their potential ability to target mRNA of factors that are known to play critical roles in eosinophil differentiation pathways or cell identify. Methodology/Principal Findings We identified 68 miRNAs with expression patterns that were up- or down- regulated 5-fold or more during bmEos differentiation. By employing TargetScan and MeSH databases, we identified 348 transcripts involved in 30 canonical pathways as potentially regulated by these miRNAs. Furthermore, by applying miRanda and Ingenuity Pathways Analysis (IPA), we identified 13 specific miRNAs that are temporally associated with the expression of IL-5Rα and CCR3 and 14 miRNAs associated with the transcription factors GATA-1/2, PU.1 and C/EBPε. We have also identified 17 miRNAs that may regulate the expression of TLRs 4 and 13 during eosinophil differentiation, although we could identify no miRNAs targeting the prominent secretory effector, eosinophil major basic protein. Conclusions/Significance This is the first study to map changes in miRNA expression in whole BM cultures during the differentiation of eosinophils, and to predict functional links between miRNAs and their target mRNAs for the regulation of eosinophilopoiesis. Our findings provide an important resource that will

  16. Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.

    PubMed

    Kumar, Parimal; Hellen, Christopher U T; Pestova, Tatyana V

    2016-07-01

    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-tRNAi (Met) 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-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l. PMID:27401559

  17. Polyadenylated and 3' processed mRNAs are transcribed from the mouse histone H2A.X gene.

    PubMed Central

    Nagata, T; Kato, T; Morita, T; Nozaki, M; Kubota, H; Yagi, H; Matsushiro, A

    1991-01-01

    We have isolated a cDNA clone encoding a mouse histone H2A.X from a cDNA library of teratocarcinoma F9 cells. The predicted amino acid sequence of this clone is 97% identical to human histone H2A.X. The first 119 residues of the mouse H2A.X were very similar (96-97%) to those of the major H2A histones (H2A.1 and H2A.2) of mouse and the long carboxy terminal sequence of H2A.X was homologous with those of several lower eukaryotes. Northern blot analysis revealed that this cDNA hybridized with two mRNAs in different sizes, 0.5 kb and 1.4 kb. The two mRNAs were present in tissue culture cells, and in spleen, thymus and testes of mice, but the ratio of abundance of the two transcripts differed in different cells and tissues. The shorter mRNA contained the highly conserved palindromic sequence typical of the 3' end of replication-dependent histone genes. The amount of this transcript was coupled to DNA synthesis and rapidly decreased in culture cells. It was synthesized just after the beginning of S-phase and degraded just after the end of S-phase. On the other hand, the longer mRNA was polyadenylated at 0.9 kb downstream from the palindromic sequence. This transcript was very stable when compared with the shorter one. These results indicate that these two mRNAs are transcribed from a single gene and maintained differently during the cell cycle, perhaps to maintain a partially replication-dependent level of histone H2A.X. Images PMID:2041781

  18. Hypothalamic Expression of Melanocortin-4 Receptor and Agouti-related Peptide mRNAs During the Estrous Cycle of Rats

    PubMed Central

    Zandi, Mohammad Reza; Jafarzadeh Shirazi, Mohammad Reza; Tamadon, Amin; Akhlaghi, Amir; Salehi, Mohammad Saied; Niazi, Ali; Moghadam, Ali

    2014-01-01

    Melanocortin- 4 receptor (MC4R) and agouti- related peptide (AgRP) are involved in energy homeostasis in rats. According to MC4R and AgRP effects on luteinizing hormone (LH) secretion, they may influence the estrous cycle of rats. Therefore, the aim of this study was to investigate the expression of MC4R and AgRP mRNAs at different stages of estrous cycle in the rat’s hypothalamus. The estrous cycle stages (proestrus, estrus, metestrus and diestrus) were determined in 20 adult female rats using vaginal smears. The rats were divided into four equal groups (n=5). Four ovariectomized rats were selected as controls two weeks after surgery. Using real- time PCR, relative expressions (compared to controls) of MC4R and AgRP mRNAs in the hypothalamus of rats were compared in four different groups of estrous cycle. The relative expression of MC4R mRNA in the hypothalamus of female rats during proestrus stage was higher than those in other stages (P=0.001). Despite a lower mean of relative expression of AgRP mRNA at proestrus stage, the relative expression of AgRP mRNA of the four stages of estrous cycle did not differ (P>0.05). In conclusion, changes in the relative expression of MC4R and AgRP mRNAs in four stages of rat estrous cycle indicated a stimulatory role of MC4R in the proestrus and preovulatory stages and an inhibitory role of AgRP in gonadotropin releasing hormone (GnRH) and LH secretions. PMID:25317405

  19. Dopamine Receptor Gene Expression in Human Amygdaloid Nuclei: Elevated D4 Receptor mRNAs in Major Depression

    PubMed Central

    Xiang, Lianbin; Szebeni, Katalin; Szebeni, Attila; Klimek, Violetta; Stockmeier, Craig A; Karolewicz, Beata; Kalbfleisch, John; Ordway, Gregory A

    2008-01-01

    Previous findings from this laboratory demonstrating changes in dopamine (DA) transporter and D2 receptors in the amygdaloid complex of subjects with major depression indicate that disruption of dopamine neurotransmission to the amygdala may contribute to behavioral symptoms associated with depression. Quantitative real-time RT-PCR was used to investigate the regional distribution of gene expression of DA receptors in the human amygdala. In addition, relative levels of mRNA of DA receptors in the basal amygdaloid nucleus were measured postmortem in subjects with major depression and normal control subjects. All five subtypes of DA receptor mRNA were detected in all amygdaloid subnuclei, although D1, D2, and D4 receptor mRNAs were more abundant than D3 and D5 mRNAs by an order of magnitude. The highest level of D1 mRNA was found in the central nucleus, whereas D2 mRNA was the most abundant in the basal nucleus. Levels of D4 mRNA were highest in the basal and central nuclei. In the basal nucleus, amounts of D4, but not D1 or D2, mRNAs were significantly higher in subjects with major depression and depressed suicide victims, as compared to control subjects. These findings demonstrate that the D1, D2 and D4 receptors are the major subtypes of DA receptors in the human amygdala. Elevated DA receptor gene expression in depressive subjects further implicates altered dopaminergic transmission in the amygdala in depression. PMID:18371940

  20. Stress and Withdrawal from Chronic Ethanol Induce Selective Changes in Neuroimmune mRNAs in Differing Brain Sites.

    PubMed

    Knapp, Darin J; Harper, Kathryn M; Whitman, Buddy A; Zimomra, Zachary; Breese, George R

    2016-01-01

    Stress is a strong risk factor in alcoholic relapse and may exert effects that mimic aspects of chronic alcohol exposure on neurobiological systems. With the neuroimmune system becoming a prominent focus in the study of the neurobiological consequences of stress, as well as chronic alcohol exposure proving to be a valuable focus in this regard, the present study sought to compare the effects of stress and chronic ethanol exposure on induction of components of the neuroimmune system. Rats were exposed to either 1 h exposure to a mild stressor (restraint) or exposure to withdrawal from 15 days of chronic alcohol exposure (i.e., withdrawal from chronic ethanol, WCE) and assessed for neuroimmune mRNAs in brain. Restraint stress alone elevated chemokine (C-C motif) ligand 2 (CCL2), interleukin-1-beta (IL-1β), tumor necrosis factor alpha (TNFα) and toll-like receptor 4 (TLR4) mRNAs in the cerebral cortex within 4 h with a return to a control level by 24 h. These increases were not accompanied by an increase in corresponding proteins. Withdrawal from WCE also elevated cytokines, but did so to varying degrees across different cytokines and brain regions. In the cortex, stress and WCE induced CCL2, TNFα, IL-1β, and TLR4 mRNAs. In the hypothalamus, only WCE induced cytokines (CCL2 and IL-1β) while in the hippocampus, WCE strongly induced CCL2 while stress and WCE induced IL-1β. In the amygdala, only WCE induced CCL2. Finally-based on the previously demonstrated role of corticotropin-releasing factor 1 (CRF1) receptor inhibition in blocking WCE-induced cytokine mRNAs-the CRF1 receptor antagonist CP154,526 was administered to a subgroup of stressed rats and found to be inactive against induction of CCL2, TNFα, or IL-1β mRNAs. These differential results suggest that stress and WCE manifest broad neuroimmune effects in brain depending on the cytokine and brain region, and that CRF inhibition may not be a relevant mechanism in non-alcohol exposed animals. Overall, these

  1. Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats.

    PubMed

    Choudhuri, Supratim; Cherrington, Nathan J; Li, Ning; Klaassen, Curtis D

    2003-11-01

    The aim of this study was to quantitatively determine the constitutive expression levels of various transporter mRNAs in rat choroid plexus. To provide a reference for the relative expression levels, the expression of various transporter mRNAs in choroid plexus were compared with that in liver, kidney, and ileum. The mRNA levels of multidrug resistance protein (Mrp)1, 2, 3, 4, 5, and 6; multidrug resistance (Mdr)1a, 1b, and 2; organic anion transporting polypeptide (Oatp)1, 2, 3, 4, 5, 9, 12, and Oat-K (1/2); organic anion transporter (Oat)1, 2, and 3; organic cation transporter (Oct)1, 2, 3, N1, and N2; bile acid transporters sodium taurocholate cotransporting polypeptide (Ntcp), bile salt excretory protein (Bsep), and ileal bile acid transporter (Ibat); divalent metal transporter 1 (DMT1), Menke's and Wilson's metal transporters; equilibrative nucleotide transporters (Ent) 1 and 2, and constitutive nucleotide transporters (Cnt)1 and 2; peptide transporters (Pept)1 and 2; as well as ATP-binding cassette (Abc)G5 and 8 were measured in choroid plexus by the branched DNA signal amplification method. Mrp1, 4, and 5, Oatp3, Menke's transporter, DMT1, Ent1, and Pept2 mRNAs were expressed in choroid plexus at higher levels than in liver, kidney, or ileum. OctN1 and N2, Oatp2, Oat2 and 3, and Cnt1 and 2 mRNAs expressions were detectable in choroid plexus, but the levels were lower compared with that in liver, kidney, or ileum. The remaining transporters [Mrp2, Mrp3, Oct1, Oct2, Oatp1, Oatp4, Oatp5, Oatp12, Oat-K (1/2), Ntcp, Bsep, Ibat, Mdr1a, Mdr1b, Mdr2, Oat1, Ent2, Pept1, AbcG5, AbcG8] were expressed at very low levels in choroid plexus. The constitutive expression levels of different transporters in choroid plexus may provide an insight into the range of xenobiotics that can potentially be transported by the choroid plexus, thereby providing a means of xenobiotic detoxification in the brain. PMID:14570765

  2. 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. PMID:25427634

  3. 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.

  4. 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

  5. Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.

    PubMed Central

    Kwong, A D; Frenkel, N

    1987-01-01

    The herpes simplex virus virion contains a function that mediates the shutoff of host-protein synthesis and the degradation of host mRNA. Viral mutants affected in this function (vhs mutants) have previously been derived. Cells infected with these mutants exhibit a more stable synthesis of host as well as the immediate early (alpha)-viral proteins. We now show that a function associated with purified virions of the wild-type virus reduces the half-life of host and alpha mRNAs, whereas purified vhs-1 mutant virions lack this activity. The functional half-life of many early (beta)- and late (gamma)-viral mRNAs is also prolonged in mutant virus infections. These studies suggest that the wild-type virion brings into cells a function that indiscriminately reduces the half-life of both host and viral transcripts and that the early translational shutoff of the host is a consequence of this function. This function may facilitate rapid transitions in the expression of groups of genes that are transcriptionally turned on at different times after infection. Images PMID:3031658

  6. PERK mediates the IRES-dependent translational activation of mRNAs encoding angiogenic growth factors after ischemic stress.

    PubMed

    Philippe, Céline; Dubrac, Alexandre; Quelen, Cathy; Desquesnes, Aurore; Van Den Berghe, Loic; Ségura, Christèle; Filleron, Thomas; Pyronnet, Stéphane; Prats, Hervé; Brousset, Pierre; Touriol, Christian

    2016-01-01

    Angiogenesis is induced by various conditions, including hypoxia. Although cap-dependent translation is globally inhibited during ischemia, the mRNAs encoding two important proangiogenic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2), are translated at early time points in ischemic muscle. The translation of these mRNAs can occur through internal ribosome entry sites (IRESs), rather than through cap-dependent translation. Hypoxic conditions also induce the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, leading us to assess the interplay between hypoxia, ER stress, and IRES-mediated translation of FGF-2 and VEGF We found that unlike cap-dependent translation, translation through FGF-2 and VEGF IRESs was efficient in cells and transgenic mice subjected to ER stress-inducing stimuli. We identified PERK, a kinase that is activated by ER stress, as the driver of VEGF and FGF-2 IRES-mediated translation in cells and in mice expressing IRES-driven reporter genes and exposed to hypoxic stress. These results demonstrate the role of IRES-dependent translation in the induction of the proangiogenic factors VEGF and FGF-2 in response to acute hypoxic stress. Furthermore, the PERK pathway could be a viable pharmacological target to improve physiological responses to ischemic situations. PMID:27141928

  7. 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

  8. TDP-43 binds and transports G-quadruplex-containing mRNAs into neurites for local translation.

    PubMed

    Ishiguro, Akira; Kimura, Nobuyuki; Watanabe, Yuto; Watanabe, Sumiko; Ishihama, Akira

    2016-05-01

    Growth and differentiation of the neurites depends on long-distance transport of a specific set of mRNAs to restricted area and their local translation. Here, we found that a TAR DNA-binding protein of 43 kDa in size (TDP-43) plays an essential role in intracellular transport of mRNA. For identification of target RNAs recognized by TDP-43, we purified TDP-43 in soluble dimer form and subjected to in vitro systematic evolution of ligands by exponential enrichment (SELEX) screening. All the TDP-43-bound RNAs were found to contain G-quadruplex (G4). Using a double-fluorescent probe system, G4-containing RNAs were found to be transported, together with TDP-43, into the distal neurites. Two lines of evidence indicated that loss of function of TDP-43 results in the neurodegenerative disorder: (i) amyotrophic lateral sclerosis (ALS)-linked mutant TDP-43M337V lacks the activity of binding and transport of G4-containing mRNAs; and (ii) RNA containing G4-forming GGGGCC repeat expansion from the ALS-linked C9orf72 gene absorbs TDP-43, thereby reducing the intracellular pool of functional TDP-43. Taken together, we propose that TDP-43 within neurons plays an essential role of mRNA transport into distal neurites for local translation, and thus, dysfunctions of TDP-43 cause neural diseases such as ALS and frontotemporal lobar degeneration. PMID:26915990

  9. Differentially expressed lncRNAs and mRNAs identified by microarray analysis in GBS patients vs healthy controls

    PubMed Central

    Xu, Jing; Gao, Chao; Zhang, Fang; Ma, Xiaofeng; Peng, Xiaolin; Zhang, Rongxin; Kong, Dexin; Simard, Alain R.; Hao, Junwei

    2016-01-01

    The aim of our present study was to determine whether message RNAs (mRNAs) and long noncoding RNAs (lncRNAs) are expressed differentially in patients with Guillain-Barré syndrome (GBS) compared with healthy controls. The mRNA and lncRNA profiles of GBS patients and healthy controls were generated by using microarray analysis. From microarray analysis, we listed 310 mRNAs and 114 lncRNAs with the mRMR software classed into two sample groups, GBS patients and healthy controls. KEGG mapping demonstrated that the top seven signal pathways may play important roles in GBS development. Several GO terms, such as cytosol, cellular macromolecular complex assembly, cell cycle, ligase activity, protein catabolic process, etc., were enriched in gene lists, suggesting a potential correlation with GBS development. Co-expression network analysis indicated that 113 lncRNAs and 303 mRNAs were included in the co-expression network. Our present study showed that these differentially expressed mRNAs and lncRNAs may play important roles in GBS development, which provides basic information for defining the mechanism(s) that promote GBS. PMID:26898505

  10. 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.