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Sample records for altered rna-binding properties

  1. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA-binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    regulating mRNA turnover in eukaryotes. However, bacterial Hfq proteins are homohexameric, whereas eukaryotic Sm/Lsm proteins are heteroheptameric. Recently, Hfq proteins with poor sequence conservation were identified in archaea and cyanobacteria. In this article, we describe crystal structures of the Hfq...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  2. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    regulating mRNA turnover in eukaryotes. However, bacterial Hfq proteins are homohexameric, whereas eukaryotic Sm/Lsm proteins are heteroheptameric. Recently, Hfq proteins with poor sequence conservation were identified in archaea and cyanobacteria. In this article, we describe crystal structures of the Hfq...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  3. Plant coilin: structural characteristics and RNA-binding properties.

    Directory of Open Access Journals (Sweden)

    Valentine Makarov

    Full Text Available Cajal bodies (CBs are dynamic subnuclear compartments involved in the biogenesis of ribonucleoproteins. Coilin is a major structural scaffolding protein necessary for CB formation, composition and activity. The predicted secondary structure of Arabidopsis thaliana coilin (Atcoilin suggests that the protein is composed of three main domains. Analysis of the physical properties of deletion mutants indicates that Atcoilin might consist of an N-terminal globular domain, a central highly disordered domain and a C-terminal domain containing a presumable Tudor-like structure adjacent to a disordered C terminus. Despite the low homology in amino acid sequences, a similar type of domain organization is likely shared by human and animal coilin proteins and coilin-like proteins of various plant species. Atcoilin is able to bind RNA effectively and in a non-specific manner. This activity is provided by three RNA-binding sites: two sets of basic amino acids in the N-terminal domain and one set in the central domain. Interaction with RNA induces the multimerization of the Atcoilin molecule, a consequence of the structural alterations in the N-terminal domain. The interaction with RNA and subsequent multimerization may facilitate coilin's function as a scaffolding protein. A model of the N-terminal domain is also proposed.

  4. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

    Science.gov (United States)

    Zhang, Jian; Lieu, Yen K; Ali, Abdullah M; Penson, Alex; Reggio, Kathryn S; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L

    2015-08-25

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

  5. Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode.

    Science.gov (United States)

    Stanek, Kimberly A; Patterson-West, Jennifer; Randolph, Peter S; Mura, Cameron

    2017-04-01

    The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore, Aae Hfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures of Aae Hfq were determined in space groups P1 and P6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

  6. Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication

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    Zeichner Steven L

    2004-12-01

    Full Text Available Abstract Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency.

  7. Prediction of altered 3'- UTR miRNA-binding sites from RNA-Seq data: the swine leukocyte antigen complex (SLA as a model region.

    Directory of Open Access Journals (Sweden)

    Marie-Laure Endale Ahanda

    Full Text Available THE SLA (swine leukocyte antigen, MHC: SLA genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3'-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs. Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1 are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered mi

  8. Prediction of altered 3'- UTR miRNA-binding sites from RNA-Seq data: the swine leukocyte antigen complex (SLA) as a model region.

    Science.gov (United States)

    Endale Ahanda, Marie-Laure; Fritz, Eric R; Estellé, Jordi; Hu, Zhi-Liang; Madsen, Ole; Groenen, Martien A M; Beraldi, Dario; Kapetanovic, Ronan; Hume, David A; Rowland, Robert R R; Lunney, Joan K; Rogel-Gaillard, Claire; Reecy, James M; Giuffra, Elisabetta

    2012-01-01

    THE SLA (swine leukocyte antigen, MHC: SLA) genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs) are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3'-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs). Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1) are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq) of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered miRNA regulation patterns.

  9. Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1.

    Science.gov (United States)

    Gershoni-Emek, Noga; Mazza, Arnon; Chein, Michael; Gradus-Pery, Tal; Xiang, Xin; Li, Ka Wan; Sharan, Roded; Perlson, Eran

    2016-02-01

    Synapse disruption takes place in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the mechanistic understanding of this process is still limited. We set out to study a possible role for dynein in synapse integrity. Cytoplasmic dynein is a multisubunit intracellular molecule responsible for diverse cellular functions, including long-distance transport of vesicles, organelles, and signaling factors toward the cell center. A less well-characterized role dynein may play is the spatial clustering and anchoring of various factors including mRNAs in distinct cellular domains such as the neuronal synapse. Here, in order to gain insight into dynein functions in synapse integrity and disruption, we performed a screen for novel dynein interactors at the synapse. Dynein immunoprecipitation from synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, followed by mass spectrometry analysis on synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, was performed. Using advanced network analysis, we identified Staufen1, an RNA-binding protein required for the transport and localization of neuronal RNAs, as a major mediator of dynein interactions via its interaction with protein phosphatase 1-beta (PP1B). Both in vitro and in vivo validation assays demonstrate the interactions of Staufen1 and PP1B with dynein, and their colocalization with synaptic markers was altered as a result of two separate ALS-linked mutations: mSOD1(G93A) and TDP43(A315T). Taken together, we suggest a model in which dynein's interaction with Staufen1 regulates mRNA localization along the axon and the synapses, and alterations in this process may correlate with synapse disruption and ALS toxicity.

  10. Novel RNA-binding properties of Pop3p support a role for eukaryotic RNase P protein subunits in substrate recognition.

    Science.gov (United States)

    Brusca, E M; True, H L; Celander, D W

    2001-11-09

    Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA molecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions (True, H. L., and Celander, D. W. (1998) J. Biol. Chem. 273, 7193-7196). We now report that Pop3p, an essential protein subunit of the holoenzyme in Saccharomyces cerevisiae, displays novel RNA-binding properties. A recombinant form of Pop3p (H6Pop3p) displays a 3-fold greater affinity for binding pre-tRNA substrates relative to tRNA products. The recognition sequence for the H6Pop3p-substrate interaction in vitro was mapped to a 39-nucleotide long sequence that extends from position -21 to +18 surrounding the natural processing site in pre-tRNA substrates. H6Pop3p binds a variety of RNA molecules with high affinity (K(d) = 16-25 nm) and displays a preference for single-stranded RNAs. Removal or modification of basic C-terminal residues attenuates the RNA-binding properties displayed by the protein specifically for a pre-tRNA substrate. These studies support the model that eukaryotic RNase P proteins bind simultaneously to the RNA subunit and RNA substrate.

  11. Engineering RNA-binding proteins for biology

    OpenAIRE

    Chen,Yu; Varani, Gabriele

    2013-01-01

    RNA-binding proteins play essential roles in the regulation of gene expression. Many have modular structures and combine relatively few common domains in various arrangements to recognize RNA sequences and/or structures. Recent progress in engineering the specificity of the PUF class RNA-binding proteins has shown that RNA-binding domains may be combined with various effector or functional domains to regulate the metabolism of targeted RNAs. Designer RNA-binding proteins with tailored sequenc...

  12. Tumor-promoting function of single nucleotide polymorphism rs1836724 (C3388T) alters multiple potential legitimate microRNA binding sites at the 3'-untranslated region of ErbB4 in breast cancer.

    Science.gov (United States)

    Bagheri, Fatemeh; Mesrian Tanha, Hamzeh; Mojtabavi Naeini, Marjan; Ghaedi, Kamran; Azadeh, Mansoureh

    2016-05-01

    ErbB4 can act as either a tumor-suppressor gene or an oncogene in breast cancer. Multiple genetic factors including single nucleotide polymorphisms (SNPs) affect gene expression patterns. Multiple 3'-untranslated region (3'-UTR) SNPs reside within the target binding site of microRNAs, which can strengthen or weaken binding to target genes. The present study aimed to predict potential 3'‑UTR variants of ErbB4 that alter the target binding site of microRNAs (miRNAs) and to clarify the association of the potential variant with the risk of developing breast cancer. In silico prediction was performed to identify potential functional SNPs within miRNA target binding sites in the 3'‑UTR of ErbB4. Thus, 146 patients and controls were genotyped using restriction fragment length polymorphism-polymerase chain reaction. In addition to the Cochran-Armitage test for trend, allele and genotype frequency differences were determined to investigate the association between rs1836724 and the susceptibility to breast cancer. Bioinformatics analysis identified rs1836724 to be a polymorphism in the seed region of four miRNA binding sites (hsa-miR335-5p, hsa-miR-28-5p, has‑miR‑708‑5p and has‑miR‑665), which may participate in the development of breast cancer. Logistic regression data indicated that the T allele of the polymorphism [OR (95% CI)=1.72 (1.056‑2.808), P=0.029] is associated with the risk of breast cancer. Using bioinformatics tools, a correlation was indicated between the presence of the T allele and a reduction in ErbB4 RNA silencing based on miRNA interaction. Furthermore, case subgroup data analysis revealed an association between the C/T genotype and an ER positive phenotype [OR (95% CI)=6.00 (1.082‑33.274), P=0.028] compared with the T/T genotype. ErbB4 and estrogen receptor 1 (ESR1) are regulated by identical miRNAs thus there may be a competition for binding sites. Due to this pattern, if the interaction between miRNAs with one gene is reduced, it

  13. PRBP: Prediction of RNA-Binding Proteins Using a Random Forest Algorithm Combined with an RNA-Binding Residue Predictor.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Xiao, Ke; Sun, Xiao

    2015-01-01

    The prediction of RNA-binding proteins is an incredibly challenging problem in computational biology. Although great progress has been made using various machine learning approaches with numerous features, the problem is still far from being solved. In this study, we attempt to predict RNA-binding proteins directly from amino acid sequences. A novel approach, PRBP predicts RNA-binding proteins using the information of predicted RNA-binding residues in conjunction with a random forest based method. For a given protein, we first predict its RNA-binding residues and then judge whether the protein binds RNA or not based on information from that prediction. If the protein cannot be identified by the information associated with its predicted RNA-binding residues, then a novel random forest predictor is used to determine if the query protein is a RNA-binding protein. We incorporated features of evolutionary information combined with physicochemical features (EIPP) and amino acid composition feature to establish the random forest predictor. Feature analysis showed that EIPP contributed the most to the prediction of RNA-binding proteins. The results also showed that the information from the RNA-binding residue prediction improved the overall performance of our RNA-binding protein prediction. It is anticipated that the PRBP method will become a useful tool for identifying RNA-binding proteins. A PRBP Web server implementation is freely available at http://www.cbi.seu.edu.cn/PRBP/.

  14. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

    NARCIS (Netherlands)

    Maatz, H.; Jens, M.; Liss, M.; Schafer, S.; Heinig, M.; Kirchner, M.; Adami, E.; Rintisch, C.; Dauksaite, V.; Radke, M.H.; Selbach, M.; Barton, P.J.; Cook, S.A.; Rajewsky, N.; Gotthardt, M.; Landthaler, M.; Hubner, N.

    2014-01-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and

  15. Poly(A) RNA-binding proteins and polyadenosine RNA: new members and novel functions.

    Science.gov (United States)

    Wigington, Callie P; Williams, Kathryn R; Meers, Michael P; Bassell, Gary J; Corbett, Anita H

    2014-01-01

    Poly(A) RNA-binding proteins (Pabs) bind with high affinity and specificity to polyadenosine RNA. Textbook models show a nuclear Pab, PABPN1, and a cytoplasmic Pab, PABPC, where the nuclear PABPN1 modulates poly(A) tail length and the cytoplasmic PABPC stabilizes poly(A) RNA in the cytoplasm and also enhances translation. While these conventional roles are critically important, the Pab family has expanded recently both in number and in function. A number of novel roles have emerged for both PAPBPN1 and PABPC that contribute to the fine-tuning of gene expression. Furthermore, as the characterization of the nucleic acid binding properties of RNA-binding proteins advances, additional proteins that show high affinity and specificity for polyadenosine RNA are being discovered. With this expansion of the Pab family comes a concomitant increase in the potential for Pabs to modulate gene expression. Further complication comes from an expansion of the potential binding sites for Pab proteins as revealed by an analysis of templated polyadenosine stretches present within the transcriptome. Thus, Pabs could influence mRNA fate and function not only by binding to the nontemplated poly(A) tail but also to internal stretches of adenosine. Understanding the diverse functions of Pab proteins is not only critical to understand how gene expression is regulated but also to understand the molecular basis for tissue-specific diseases that occur when Pab proteins are altered. Here we describe both conventional and recently emerged functions for PABPN1 and PABPC and then introduce and discuss three new Pab family members, ZC3H14, hnRNP-Q1, and LARP4.

  16. RNA-binding proteins in mouse male germline stem cells: a mammalian perspective.

    Science.gov (United States)

    Qi, Huayu

    2016-01-01

    Adult stem cells that reside in particular types of tissues are responsible for tissue homeostasis and regeneration. Cellular functions of adult stem cells are intricately related to the gene expression programs in those cells. Past research has demonstrated that regulation of gene expression at the transcriptional level can decisively alter cell fate of stem cells. However, cellular contents of mRNAs are sometimes not equivalent to proteins, the functional units of cells. It is increasingly realized that post-transcriptional and translational regulation of gene expression are also fundamental for stem cell functions. Compared to differentiated somatic cells, effects on cellular status manifested by varied expression of RNA-binding proteins and global protein synthesis have been demonstrated in several stem cell systems. Through the cooperation of both cis-elements of mRNAs and trans-acting RNA-binding proteins that are intimately associated with them, regulation of localization, stability, and translational status of mRNAs directly influences the self-renewal and differentiation of stem cells. Previous studies have uncovered some of the molecular mechanisms that underlie the functions of RNA-binding proteins in stem cells in invertebrate species. However, their roles in adult stem cells in mammals are just beginning to be unveiled. This review highlights some of the RNA-binding proteins that play important functions during the maintenance and differentiation of mouse male germline stem cells, the adult stem cells in the male reproductive organ.

  17. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

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    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  18. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    Science.gov (United States)

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

  19. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  20. Modulation of RNase E activity by alternative RNA binding sites.

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    Daeyoung Kim

    Full Text Available Endoribonuclease E (RNase E affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37 of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13 containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433, whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71. Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

  1. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  2. Hacking RNA: Hakai promotes tumorigenesis by switching on the RNA-binding function of PSF

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    Figueroa, Angélica; Fujita, Yasuyuki; Gorospe, Myriam

    2009-01-01

    Hakai, an E3 ubiquitin ligase for the E-cadherin complex, plays a crucial role in lowering cell-cell contacts in epithelial cells, a hallmark feature of tumor progression. Recently, Hakai was also found to interact with PSF (PTB-associated splicing factor). While PSF can function as a DNA-binding protein with a tumor suppressive function, its association with Hakai promotes PSF’s RNA-binding ability and post-transcriptional influence on target mRNAs. Hakai overexpression enhanced the binding of PSF to mRNAs encoding cancer-related proteins, while knockdown of Hakai reduced the RNA-binding ability of PSF. Furthermore, the knockdown of PSF suppressed Hakai-induced cell proliferation. Thus, Hakai can affect the oncogenic phenotype both by altering E-cadherin-based intercellular adhesions and by increasing PSF’s ability to bind RNAs that promote cancer-related gene expression. PMID:19855157

  3. Hacking RNA: Hakai promotes tumorigenesis by enhancing the RNA-binding function of PSF.

    Science.gov (United States)

    Figueroa, Angélica; Fujita, Yasuyuki; Gorospe, Myriam

    2009-11-15

    Hakai, an E3 ubiquitin ligase for the E-cadherin complex, plays a crucial role in lowering cell-cell contacts in epithelial cells, a hallmark feature of tumor progression. Recently, Hakai was also found to interact with PSF (PTB-associated splicing factor). While PSF can function as a DNA-binding protein with a tumor suppressive function, its association with Hakai promotes PSF's RNA-binding ability and post-transcriptional influence on target mRNAs. Hakai overexpression enhanced the binding of PSF to mRNAs encoding cancer-related proteins, while knockdown of Hakai reduced the RNA-binding ability of PSF. Furthermore, the knockdown of PSF suppressed Hakai-induced cell proliferation. Thus, Hakai can affect the oncogenic phenotype both by altering E-cadherin-based intercellular adhesions and by increasing PSF's ability to bind RNAs that promote cancer-related gene expression.

  4. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

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    Elisa E. Figueroa-Angulo

    2015-11-01

    Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  5. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

    Science.gov (United States)

    Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-11-26

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  6. Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.

    Directory of Open Access Journals (Sweden)

    Yann Saint-Georges

    Full Text Available The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators. Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings

  7. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-08

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner.

  8. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  9. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Science.gov (United States)

    Hogan, Gregory J; Brown, Patrick O; Herschlag, Daniel

    2015-01-01

    Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1) Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2) In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa) in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae) suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3) The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA) cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4) Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron transport

  10. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Directory of Open Access Journals (Sweden)

    Gregory J Hogan

    Full Text Available Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1 Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2 In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3 The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4 Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron

  11. Plant RNA binding proteins for control of RNA virus infection

    OpenAIRE

    Huh, Sung Un; Paek, Kyung-Hee

    2013-01-01

    Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific b...

  12. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  13. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  14. Pumilio Puf domain RNA-binding proteins in Arabidopsis.

    Science.gov (United States)

    Abbasi, Nazia; Park, Youn-Il; Choi, Sang-Bong

    2011-03-01

    Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35-39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such as humans and plants. In yeast and animals, they are involved in a variety of posttranscriptional RNA metabolism including RNA decay, RNA transport, rRNA processing and translational repression. However, their roles in plants are largely unknown. Recently, we have characterized the first member of the Puf family of RNA-binding proteins, APUM23, in Arabidopsis. Here, we discuss and summarize the diverse roles and targets of Puf proteins previously reported in other organisms and then highlight the potential regulatory roles of Puf proteins in Arabidopsis, using our recent study as an example.

  15. CAG trinucleotide RNA repeats interact with RNA-binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    McLaughlin, B.A.; Eberwine, J.; Spencer, C. [Univ. of Pennsylvania, Philadelphia, PA (United States)

    1996-09-01

    Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington`s disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to >37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and LJV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases. 47 refs., 5 figs.

  16. RNA-binding proteins in microsatellite expansion disorders: mediators of RNA toxicity.

    Science.gov (United States)

    Echeverria, Gloria V; Cooper, Thomas A

    2012-06-26

    Although protein-mediated toxicity in neurological disease has been extensively characterized, RNA-mediated toxicity is an emerging mechanism of pathogenesis. In microsatellite expansion disorders, expansion of repeated sequences in noncoding regions gives rise to RNA that produces a toxic gain of function, while expansions in coding regions can disrupt protein function as well as produce toxic RNA. The toxic RNA typically aggregates into nuclear foci and contributes to disease pathogenesis. In many cases, toxicity of the RNA is caused by the disrupted functions of RNA-binding proteins. We will discuss evidence for RNA-mediated toxicity in microsatellite expansion disorders. Different microsatellite expansion disorders are linked with alterations in the same as well as disease-specific RNA-binding proteins. Recent studies have shown that microsatellite expansions can encode multiple repeat-containing toxic RNAs through bidirectional transcription and protein species through repeat-associated non-ATG translation. We will discuss approaches that have characterized the toxic contributions of these various factors.

  17. RNA-binding protein CPEB1 remodels host and viral RNA landscapes.

    Science.gov (United States)

    Batra, Ranjan; Stark, Thomas J; Clark, Elizabeth; Belzile, Jean-Philippe; Wheeler, Emily C; Yee, Brian A; Huang, Hui; Gelboin-Burkhart, Chelsea; Huelga, Stephanie C; Aigner, Stefan; Roberts, Brett T; Bos, Tomas J; Sathe, Shashank; Donohue, John Paul; Rigo, Frank; Ares, Manuel; Spector, Deborah H; Yeo, Gene W

    2016-12-01

    Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.

  18. Single nucleotide polymorphisms in microRNA binding sites of oncogenes: implications in cancer and pharmacogenomics.

    Science.gov (United States)

    Manikandan, Mayakannan; Munirajan, Arasambattu Kannan

    2014-02-01

    Cancer, a complex genetic disease involving uncontrolled cell proliferation, is caused by inactivation of tumor suppressor genes and activation of oncogenes. A vast majority of these cancer causing genes are known targets of microRNAs (miRNAs) that bind to complementary sequences in 3' untranslated regions (UTR) of messenger RNAs and repress them from translation. Single Nucleotide Polymorphisms (SNPs) occurring naturally in such miRNA binding regions can alter the miRNA:mRNA interaction and can significantly affect gene expression. We hypothesized that 3'UTR SNPs in miRNA binding sites of proto-oncogenes could abrogate their post-transcriptional regulation, resulting in overexpression of oncogenic proteins, tumor initiation, progression, and modulation of drug response in cancer patients. Therefore, we developed a systematic computational pipeline that integrates data from well-established databases, followed stringent selection criteria and identified a panel of 30 high-confidence SNPs that may impair miRNA target sites in the 3' UTR of 54 mRNA transcripts of 24 proto-oncogenes. Further, 8 SNPs amidst them had the potential to determine therapeutic outcome in cancer patients. Functional annotation suggested that altogether these SNPs occur in proto-oncogenes enriched for kinase activities. We provide detailed in silico evidence for the functional effect of these candidate SNPs in various types of cancer.

  19. RNA binding protein Pub1p regulates glycerol production and stress tolerance by controlling Gpd1p activity during winemaking.

    Science.gov (United States)

    Orozco, Helena; Sepúlveda, Ana; Picazo, Cecilia; Matallana, Emilia; Aranda, Agustín

    2016-06-01

    Glycerol is a key yeast metabolite in winemaking because it contributes to improve the organoleptic properties of wine. It is also a cellular protective molecule that enhances the tolerance of yeasts to osmotic stress and promotes longevity. Thus, its production increases by genetic manipulation, which is of biotechnological and basic interest. Glycerol is produced by diverting glycolytic glyceraldehyde-3-phosphate through the action of glycerol-3-phosphate dehydrogenase (coded by genes GPD1 and GPD2). Here, we demonstrate that RNA-binding protein Pub1p regulates glycerol production by controlling Gpd1p activity. Its deletion does not alter GPD1 mRNA levels, but protein levels and enzymatic activity increase, which explains the higher intracellular glycerol concentration and greater tolerance to osmotic stress of the pub1∆ mutant. PUB1 deletion also enhances the activity of nicotinamidase, a longevity-promoting enzyme. Both enzymatic activities are partially located in peroxisomes, and we detected peroxisome formation during wine fermentation. The role of Pub1p in life span control depends on nutrient conditions and is related with the TOR pathway, and a major connection between RNA metabolism and the nutrient signaling response is established.

  20. RNA-binding protein Lin28 in cancer and immunity.

    Science.gov (United States)

    Jiang, Shuai; Baltimore, David

    2016-05-28

    The highly conserved RNA-binding protein, Lin28, is involved in many biological processes, including development, reprogramming, pluripotency, and metabolism. Importantly, Lin28 functions as an oncogene, promoting tumor progression and metastasis in various human cancers. Lin28 can regulate gene expression either by directly binding to mRNAs or by blocking microRNA biogenesis, and the underlying mechanisms include Let-7-dependent and Let-7-independent modes of action. Recent evidence shows that Lin28 also plays a fundamental role in immunity. The roles of Lin28 in disease are complex and require characterization of its physiological functions in cancer and immunological contexts. Here we review emerging information on the role of Lin28 in cancer and immunity and the molecular mechanisms it uses. We discuss our present knowledge of the system and highlight remaining mysteries related to the functions of this small RNA-binding protein. This knowledge may lead to Lin28 becoming a diagnostic marker for cancer or immune-related diseases and a possible therapeutic target.

  1. From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces.

    Science.gov (United States)

    Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

    2011-09-01

    Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design.

  2. Dynamic nucleocytoplasmic shuttling of an Arabidopsis SR splicing factor: role of the RNA-binding domains.

    Science.gov (United States)

    Rausin, Glwadys; Tillemans, Vinciane; Stankovic, Nancy; Hanikenne, Marc; Motte, Patrick

    2010-05-01

    Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors. We have investigated the dynamic subcellular distribution of the Arabidopsis (Arabidopsis thaliana) RSZp22 protein, a homolog of the human 9G8 SR factor. Little is known about the determinants underlying the control of plant SR protein dynamics, and so far most studies relied on ectopic transient overexpression. Here, we provide a detailed analysis of the RSZp22 expression profile and describe its nucleocytoplasmic shuttling properties in specific cell types. Comparison of transient ectopic- and stable tissue-specific expression highlights the advantages of both approaches for nuclear protein dynamic studies. By site-directed mutagenesis of RSZp22 RNA-binding sequences, we show that functional RNA recognition motif RNP1 and zinc-knuckle are dispensable for the exclusive protein nuclear localization and speckle-like distribution. Fluorescence resonance energy transfer imaging also revealed that these motifs are implicated in RSZp22 molecular interactions. Furthermore, the RNA-binding motif mutants are defective for their export through the CRM1/XPO1/Exportin-1 receptor pathway but retain nucleocytoplasmic mobility. Moreover, our data suggest that CRM1 is a putative export receptor for mRNPs in plants.

  3. RNA binding efficacy of theophylline, theobromine and caffeine.

    Science.gov (United States)

    Johnson, I Maria; Kumar, S G Bhuvan; Malathi, R

    2003-04-01

    The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.

  4. Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity

    Directory of Open Access Journals (Sweden)

    Zachary T. Campbell

    2012-05-01

    Full Text Available The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach, we analyzed members of the PUF (Pumilio and FBF family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.

  5. Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins.

    Science.gov (United States)

    Castello, Alfredo; Hentze, Matthias W; Preiss, Thomas

    2015-12-01

    In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve 'metabolic remodeling' with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation ('moonlighting') and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme-RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future.

  6. RNA-binding proteins in plants: the tip of an iceberg?

    Science.gov (United States)

    Fedoroff, Nina V.; Federoff, N. V. (Principal Investigator)

    2002-01-01

    RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.

  7. The Arabidopsis RNA-Binding Protein AtRGGA Regulates Tolerance to Salt and Drought Stress

    KAUST Repository

    Ambrosone, Alfredo

    2015-03-17

    Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

  8. Sequence-Based Prediction of RNA-Binding Proteins Using Random Forest with Minimum Redundancy Maximum Relevance Feature Selection

    Directory of Open Access Journals (Sweden)

    Xin Ma

    2015-01-01

    Full Text Available The prediction of RNA-binding proteins is one of the most challenging problems in computation biology. Although some studies have investigated this problem, the accuracy of prediction is still not sufficient. In this study, a highly accurate method was developed to predict RNA-binding proteins from amino acid sequences using random forests with the minimum redundancy maximum relevance (mRMR method, followed by incremental feature selection (IFS. We incorporated features of conjoint triad features and three novel features: binding propensity (BP, nonbinding propensity (NBP, and evolutionary information combined with physicochemical properties (EIPP. The results showed that these novel features have important roles in improving the performance of the predictor. Using the mRMR-IFS method, our predictor achieved the best performance (86.62% accuracy and 0.737 Matthews correlation coefficient. High prediction accuracy and successful prediction performance suggested that our method can be a useful approach to identify RNA-binding proteins from sequence information.

  9. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    Science.gov (United States)

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

  10. Protein universe containing a PUA RNA-binding domain.

    Science.gov (United States)

    Cerrudo, Carolina S; Ghiringhelli, Pablo D; Gomez, Daniel E

    2014-01-01

    Here, we review current knowledge about pseudouridine synthase and archaeosine transglycosylase (PUA)-domain-containing proteins to illustrate progress in this field. A methodological analysis of the literature about the topic was carried out, together with a 'qualitative comparative analysis' to give a more comprehensive review. Bioinformatics methods for whole-protein or protein-domain identification are commonly based on pairwise protein sequence comparisons; we added comparison of structures to detect the whole universe of proteins containing the PUA domain. We present an update of proteins having this domain, focusing on the specific proteins present in Homo sapiens (dyskerin, MCT1, Nip7, eIF2D and Nsun6), and explore the existence of these in other species. We also analyze the phylogenetic distribution of the PUA domain in different species and proteins. Finally, we performed a structural comparison of the PUA domain through data mining of structural databases, determining a conserved structural motif, despite the differences in the sequence, even among eukaryotes, archaea and bacteria. All data discussed in this review, both bibliographic and analytical, corroborate the functional importance of the PUA domain in RNA-binding proteins.

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

    OpenAIRE

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

    2009-01-01

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

  12. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

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    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  13. RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp.

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    Chumporn Soowannayan

    Full Text Available Gill-associated virus (GAV infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs using His(6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ssRNAs tested independent of their sequence. The ssRNAs included (+ and (- sense regions of the GAV genome as well as a (+ sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (dsRNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa N-terminal sequence spanning Met(11 to Arg(60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11PVRRPLPPQPPRNARLI(29 encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.

  14. Regulation of RNA binding proteins in trypanosomatid protozoan parasites.

    Science.gov (United States)

    Romaniuk, María Albertina; Cervini, Gabriela; Cassola, Alejandro

    2016-02-26

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

  15. Regulation of RNA binding proteins in trypanosomatid protozoan parasites

    Institute of Scientific and Technical Information of China (English)

    María Albertina Romaniuk; Gabriela Cervini; Alejandro Cassola

    2016-01-01

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins(RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of m RNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

  16. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

    Directory of Open Access Journals (Sweden)

    Sebastien Lageix

    2015-02-01

    Full Text Available The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype, while other substitutions block kinase activation (Gcn- phenotype, in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD, previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

  17. Roles of RNA-Binding Proteins in DNA Damage Response

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    Mihoko Kai

    2016-02-01

    Full Text Available Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR, and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP with low complexity domains, called intrinsically disordered proteins (IDPs, and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs in a poly(ADP-ribose (PAR-dependent manner (unpublished data. DNA-dependent PARP1 (poly-(ADP ribose polymerase 1 makes key contributions in the DNA damage response (DDR network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  18. Genome-Wide microRNA Binding Site Variation between Extinct Wild Aurochs and Modern Cattle Identifies Candidate microRNA-Regulated Domestication Genes

    Science.gov (United States)

    Braud, Martin; Magee, David A.; Park, Stephen D. E.; Sonstegard, Tad S.; Waters, Sinead M.; MacHugh, David E.; Spillane, Charles

    2017-01-01

    The domestication of cattle from the now-extinct wild aurochs (Bos primigenius) involved selection for physiological and behavioral traits, with underlying genetic factors that remain largely unknown. Non-coding microRNAs have emerged as key regulators of the spatio-temporal expression of target genes controlling mammalian growth and development, including in livestock species. During the domestication process, selection of mutational changes in miRNAs and/or miRNA binding sites could have provided a mechanism to generate some of the traits that differentiate domesticated cattle from wild aurochs. To investigate this, we analyzed the open reading frame DNA sequence of 19,994 orthologous protein-coding gene pairs from extant Bos taurus genomes and a single extinct B. primigenius genome. We identified miRNA binding site polymorphisms in the 3′ UTRs of 1,620 of these orthologous genes. These 1,620 genes with altered miRNA binding sites between the B. taurus and B. primigenius lineages represent candidate domestication genes. Using a novel Score Site ratio metric we have ranked these miRNA-regulated genes according to the extent of divergence between miRNA binding site presence, frequency and copy number between the orthologous genes from B. taurus and B. primigenius. This provides an unbiased approach to identify cattle genes that have undergone the most changes in miRNA binding (i.e., regulation) between the wild aurochs and modern-day cattle breeds. In addition, we demonstrate that these 1,620 candidate domestication genes are enriched for roles in pigmentation, fertility, neurobiology, metabolism, immunity and production traits (including milk quality and feed efficiency). Our findings suggest that directional selection of miRNA regulatory variants was important in the domestication and subsequent artificial selection that gave rise to modern taurine cattle. PMID:28197171

  19. RNA-binding proteins related to stress response and differentiation in protozoa.

    Science.gov (United States)

    Alves, Lysangela Ronalte; Goldenberg, Samuel

    2016-02-26

    RNA-binding proteins (RBPs) are key regulators of gene expression. There are several distinct families of RBPs and they are involved in the cellular response to environmental changes, cell differentiation and cell death. The RBPs can differentially combine with RNA molecules and form ribonucleoprotein (RNP) complexes, defining the function and fate of RNA molecules in the cell. RBPs display diverse domains that allow them to be categorized into distinct families. They play important roles in the cellular response to physiological stress, in cell differentiation, and, it is believed, in the cellular localization of certain mRNAs. In several protozoa, a physiological stress (nutritional, temperature or pH) triggers differentiation to a distinct developmental stage. Most of the RBPs characterized in protozoa arise from trypanosomatids. In these protozoa gene expression regulation is mostly post-transcriptional, which suggests that some RBPs might display regulatory functions distinct from those described for other eukaryotes. mRNA stability can be altered as a response to stress. Transcripts are sequestered to RNA granules that ultimately modulate their availability to the translation machinery, storage or degradation, depending on the associated proteins. These aggregates of mRNPs containing mRNAs that are not being translated colocalize in cytoplasmic foci, and their numbers and size vary according to cell conditions such as oxidative stress, nutritional status and treatment with drugs that inhibit translation.

  20. RNA-binding proteins related to stress response and differentiation in protozoa

    Institute of Scientific and Technical Information of China (English)

    Lysangela Ronalte Alves; Samuel Goldenberg

    2016-01-01

    RNA-binding proteins(RBPs) are key regulators of gene expression. There are several distinct families of RBPs and they are involved in the cellular response to environmental changes, cell differentiation and cell death. The RBPs can differentially combine with RNA molecules and form ribonucleoprotein(RNP) complexes, defining the function and fate of RNA molecules in the cell. RBPs display diverse domains that allow them to be categorized into distinct families. They play important roles in the cellular response to physiological stress, in cell differentiation, and, it is believed, in the cellular localization of certain mRNAs. In several protozoa, a physiological stress(nutritional, temperature or pH) triggers differentiation to a distinct developmental stage. Most of the RBPs characterized in protozoa arise from trypanosomatids. In these protozoa gene expression regulation is mostly post-transcriptional, which suggests that some RBPs might display regulatory functions distinct from those described for other eukaryotes. mRNA stability can be altered as a response to stress. Transcripts are sequestered to RNA granules that ultimately modulate their availability to the translation machinery, storage or degradation, depending on the associated proteins. These aggregates of mRNPs containing mRNAs that are not being translated colocalize in cytoplasmic foci, and their numbers and size vary according to cell conditions such as oxidative stress, nutritional status and treatment with drugs that inhibit translation.

  1. SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

    Science.gov (United States)

    Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

    2016-10-20

    RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.

  2. Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

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    Brittney R Henderson

    Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

  3. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

  4. UPF201 Archaeal Specific Family Members Reveals Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.N.; Swaminathan, S.; Burley, S. K.

    2008-12-11

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  5. UPF201 Archaeal Specific Family Members Reveal Structural Similarity to RNA-Binding Proteins but Low Likelyhood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.; Burley, S; Swaminathan, S

    2008-01-01

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  6. UPF201 archaeal specific family members reveal structural similarity to RNA-binding proteins but low likelihood for RNA-binding function.

    Directory of Open Access Journals (Sweden)

    Krishnamurthy N Rao

    Full Text Available We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54 to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40% and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel beta-sheet and five alpha-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  7. Control of signaling in a MAP-kinase pathway by an RNA-binding protein.

    Directory of Open Access Journals (Sweden)

    Susanne Prinz

    Full Text Available Signaling-protein mRNAs tend to have long untranslated regions (UTRs containing binding sites for RNA-binding proteins regulating gene expression. Here we show that a PUF-family RNA-binding protein, Mpt5, represses the yeast MAP-kinase pathway controlling differentiation to the filamentous form. Mpt5 represses the protein levels of two pathway components, the Ste7 MAP-kinase kinase and the Tec1 transcriptional activator, and negatively regulates the kinase activity of the Kss1 MAP kinase. Moreover, Mpt5 specifically inhibits the output of the pathway in the absence of stimuli, and thereby prevents inappropriate cell differentiation. The results provide an example of what may be a genome-scale level of regulation at the interface of signaling networks and protein-RNA binding networks.

  8. Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis.

    Science.gov (United States)

    Zhang, Hai-Tao; Xue, Jing-Hui; Zhang, Zhi-Wen; Kong, Hai-Bo; Liu, Ai-Jun; Li, Shou-Chun; Xu, Dong-Gang

    2015-10-01

    Cold-inducible RNA-binding protein (CIRP) is induced by mild hypothermia in several mammals, but the precise mechanism by which CIRP mediates hypothermia-induced neuroprotection remains unknown. We aimed to investigate the molecular mechanisms by which CIRP protects the nervous system during mild hypothermia. Rat cortical neurons were isolated and cultured in vitro under mild hypothermia (32°C). Apoptosis was measured by annexin V and propidium iodide staining, visualized by flow cytometry. Neuron ultrastructure was visualized by transmission electron microscopy. CIRP overexpression and knockdown were achieved via infection with pL/IRES/GFP-CIRP and pL/shRNA/F-CIRP-A lentivirus. RT(2) Profiler PCR Array Pathway Analysis and western blotting were used to evaluate the effects of CIRP overexpresion/knockdown on the neurons׳ transcriptome. Neuron late apoptosis was significantly reduced at day 7 of culture by 12h hypothermia, but neuron ultrastructure remained relatively intact. RT(2) Profiler PCR Array Pathway Analysis of 84 apoptosis pathway-associated factors revealed that mild hypothermia and CIRP overexpression induce similar gene expression profiles, specifically alterations of genes implicated in the mitochondrial apoptosis pathway. Mild hypothermia-treated neurons up-regulated 12 and down-regulated 38 apoptosis pathway-associated genes. CIRP-overexpressing neurons up-regulated 15 and down-regulated 46 genes. CIRP-knocked-down hypothermia-treated cells up-regulated 9 and down-regulated 40 genes. Similar results were obtained at the protein level. In conclusion, CIRP may inhibit neuron apoptosis through the suppression of the mitochondria apoptosis pathway during mild hypothermia.

  9. A versatile assay for RNA-binding proteins in living cells.

    Science.gov (United States)

    Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W; Castello, Alfredo

    2014-05-01

    RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.

  10. Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins

    OpenAIRE

    Borgeson, Claudia D.; Samson, Marie-Laure

    2005-01-01

    The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3′-untranslated region (3′-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3′-UTR. This site resembles ELAV-binding sites identified prev...

  11. Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements.

    Directory of Open Access Journals (Sweden)

    Amanda Swain

    2016-09-01

    Full Text Available The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs. RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.

  12. Interacting protein partners of Arabidopsis RNA binding protein AtRBP45b

    Science.gov (United States)

    RNA binding proteins (RBPs) are important players in post-transcriptional gene regulation and shown to play an important role in normal development and in response to environmental perturbations. Arabidopsis RBP, AtRBP45b with triple RNA recognition motifs (RRMs) have are closely related to the yeas...

  13. STarMir Tools for Prediction of microRNA binding sites

    Science.gov (United States)

    Kanoria, Shaveta; Rennie, William; Liu, Chaochun; Carmack, C. Steven; Lu, Jun; Ding, Ye

    2017-01-01

    MicroRNAs (miRNAs) are a class of endogenous short non-coding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs), which results in translational repression and/or mRNA degradation. As regulatory molecules, miRNAs are involved in many mammalian biological processes and also in the manifestation of certain human diseases. As miRNAs play central role in the regulation of gene expression, understanding miRNA-binding patterns is essential to gain an insight of miRNA mediated gene regulation and also holds promise for therapeutic applications. Computational prediction of miRNA binding sites on target mRNAs facilitates experimental investigation of miRNA functions. This chapter provides protocols for using the STarMir web server for improved predictions of miRNA binding sites on a target mRNA. As an application module of the Sfold RNA package, the current version of STarMir is an implementation of logistic prediction models developed with high throughput miRNA binding data from crosslinking immuno-precipitation (CLIP) studies. The models incorporated comprehensive thermodynamic, structural and sequence features, and were found to make improved predictions of both seed and seedless sites, in comparison to the established algorithms [1]. Their broad applicability was indicated by their good performance in cross-species validation. STarMir is freely available at http://sfold.wadsworth.org/starmir.html PMID:27665594

  14. SCA31 Flies Perform in a Balancing Act between RAN Translation and RNA-Binding Proteins.

    Science.gov (United States)

    Jackson, George R

    2017-04-05

    In this issue of Neuron, Ishiguro et al. (2017) explore the toxicity of RAN translation in spinocerebellar ataxia 31. Using a Drosophila model, the authors demonstrate that TDP-43 and other RNA-binding proteins act as chaperones to regulate the formation of toxic RNA aggregates.

  15. A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence.

    Science.gov (United States)

    Zhang, Chi; Muench, Douglas G

    2015-12-11

    PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5' end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.

  16. Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

    Science.gov (United States)

    Qiu, Chen; Kershner, Aaron; Wang, Yeming; Holley, Cynthia P; Wilinski, Daniel; Keles, Sunduz; Kimble, Judith; Wickens, Marvin; Hall, Traci M Tanaka

    2012-02-24

    mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.

  17. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

    Science.gov (United States)

    Kash, J C; Menon, K M

    1999-12-21

    In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

  18. Characterization of RNA-Protein Interactions: Lessons from Two RNA-Binding Proteins, SRSF1 and SRSF2.

    Science.gov (United States)

    Skrdlant, Lindsey; Lin, Ren-Jang

    2016-01-01

    SR proteins are a class of RNA-binding proteins whose RNA-binding ability is required for both constitutive and alternative splicing. While members of the SR protein family were once thought to have redundant functions, in-depth biochemical analysis of their RNA-binding abilities has revealed distinct binding profiles for each SR protein, that often lead to either synergistic or antagonistic functions. SR protein family members SRSF1 and SRSF2 are two of the most highly studied RNA-binding proteins. Here we examine the various methods used to differentiate SRSF1 and SRSF2 RNA-binding ability. We discuss the benefits and type of information that can be determined using each method.

  19. Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

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    Changrui Lu

    Full Text Available BACKGROUND: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. METHODOLOGY/PRINCIPAL FINDINGS: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. CONCLUSION/SIGNIFICANCE: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

  20. Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei.

    Science.gov (United States)

    Kafková, Lucie; Ammerman, Michelle L; Faktorová, Drahomíra; Fisk, John C; Zimmer, Sara L; Sobotka, Roman; Read, Laurie K; Lukes, Julius; Hashimi, Hassan

    2012-10-01

    A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

  1. The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

    Science.gov (United States)

    Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

    2013-09-13

    MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

  2. Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Thiruselvam, Viswanathan; Sivaraman, Padavattan; Kumarevel, Thirumananseri; Ponnuswamy, Mondikalipudur Nanjappagounder

    2014-04-18

    RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP-l-histidine-Mg(2+)) and EDTA (0.5 M) treated ternary complex (HutP-l-histidine-Mg(2+)), solved at 1.9 Å and 2.5 Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5 M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.

  3. False positive RNA binding activities after Ni-affinity purification from Escherichia coli.

    Science.gov (United States)

    Milojevic, Tetyana; Sonnleitner, Elisabeth; Romeo, Alessandra; Djinović-Carugo, Kristina; Bläsi, Udo

    2013-06-01

    A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

  4. Phloem RNA-binding proteins as potential components of the long-distance RNA transport system.

    Directory of Open Access Journals (Sweden)

    VICENTE ePALLAS

    2013-05-01

    Full Text Available RNA-binding proteins (RBPs govern a myriad of different essential processes in eukaryotic cells. Recent evidence reveals that apart from playing critical roles in RNA metabolism and RNA transport, RBPs perform a key function in plant adaption to various environmental conditions. Long distance RNA transport occurs in land plants through the phloem, a conducting tissue that integrates the wide range of signalling pathways required to regulate plant development and response to stress processes. The macromolecules in the phloem pathway vary greatly and include defence proteins, transcription factors, chaperones acting in long distance trafficking, and RNAs (mRNAs, siRNAs and miRNAs. How these RNA molecules translocate through the phloem is not well understood, but recent evidence indicates the presence of translocatable RNA-binding proteins in the phloem, which act as potential components of long distance RNA transport system. This review updates our knowledge on the characteristics and functions of RBPs present in the phloem.

  5. RNA-binding protein hnRNPLL as a critical regulator of lymphocyte homeostasis and differentiation.

    Science.gov (United States)

    Chang, Xing

    2016-05-01

    RNA-binding proteins orchestrate posttranscriptional regulation of gene expression, such as messenger RNA (mRNA) splicing, RNA stability regulation, and translation regulation. Heterogeneous nuclear RNA-binding proteins (hnRNPs) refer to a collection of unrelated RNA-binding proteins predominantly located in the nucleus (Han et al. Biochem J 2010, 430:379-392). Although canonical functions of hnRNPs are to promote pre-mRNA splicing, they are involved in all the processes of RNA metabolism through recognizing specific cis-elements on RNA (Dreyfuss et al. Annu Rev Biochem 1993, 62:289-321; Huelga et al. Cell Rep 2012, 1:167-178; Krecic and Swanson. Curr Opin Cell Biol 1999, 11:363-371). Heterogeneous nuclear RNA-binding protein L like (hnRNPLL) is a tissue-specific hnRNP, which was identified as a regulator of CD45RA to CD45RO switching during memory T-cell development (Oberdoerffer et al. Science 2008, 321:686-691; Topp et al. RNA 2008, 14:2038-2049; Wu et al. Immunity 2008, 29:863-875). Since then, hnRNPLL has emerged as a critical regulator of lymphocyte homeostasis and terminal differentiation, controlling alternative splicing or expression of critical genes for the lymphocytes development (Wu et al. Immunity 2008, 29:863-875; Chang et al. Proc Natl Acad Sci USA 2015, 112:E1888-E1897). This review will summarize recent advances in understanding the functions of hnRNPLL, focusing on its biochemical functions and physiological roles in lymphocyte differentiation and homeostasis. WIREs RNA 2016, 7:295-302. doi: 10.1002/wrna.1335 For further resources related to this article, please visit the WIREs website.

  6. Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

    OpenAIRE

    Yacine Kharraz; Pierre-Adrien Salmand; Anne Camus; Jacques Auriol; Cyril Gueydan; Véronique Kruys; Dominique Morello

    2010-01-01

    BACKGROUND: TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translationa...

  7. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

  8. Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R.

    Science.gov (United States)

    Kraushar, Matthew L; Thompson, Kevin; Wijeratne, H R Sagara; Viljetic, Barbara; Sakers, Kristina; Marson, Justin W; Kontoyiannis, Dimitris L; Buyske, Steven; Hart, Ronald P; Rasin, Mladen-Roko

    2014-09-09

    Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

  9. The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

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    Daniel Larocque

    Full Text Available BACKGROUND: The quaking viable (qk(v mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS. The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination. METHODOLOGY/PRINCIPAL FINDINGS: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1 and myelin basic protein (MBP, markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

  10. RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation.

    Science.gov (United States)

    Palanichamy, Jayanth Kumar; Tran, Tiffany M; Howard, Jonathan M; Contreras, Jorge R; Fernando, Thilini R; Sterne-Weiler, Timothy; Katzman, Sol; Toloue, Masoud; Yan, Weihong; Basso, Giuseppe; Pigazzi, Martina; Sanford, Jeremy R; Rao, Dinesh S

    2016-04-01

    Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia-rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3' untranslated regions (3'UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

  11. Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

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    Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

    2014-09-26

    Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

  12. Altered plasma fibrin clot properties in essential thrombocythemia.

    Science.gov (United States)

    Małecki, Rafał; Gacka, Małgorzata; Kuliszkiewicz-Janus, Małgorzata; Jakobsche-Policht, Urszula; Kwiatkowski, Jacek; Adamiec, Rajmund; Undas, Anetta

    2016-01-01

    Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (Ks), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991 × 10(3)/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced Ks (-38%, p Ks inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.

  13. The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease

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    Yalin Liao

    2016-08-01

    Full Text Available RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism.

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

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    Scott eTenenbaum

    2014-04-01

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

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

    Science.gov (United States)

    Xin, HongBo; Deng, KeYu; Fu, MinGui

    2014-08-01

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

  16. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

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    David Piñeiro

    2015-04-01

    Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

  17. Structural delineation of stem-loop RNA binding by human TAF15 protein.

    Science.gov (United States)

    Kashyap, Maruthi; Ganguly, Akshay Kumar; Bhavesh, Neel Sarovar

    2015-11-27

    Human TATA binding protein associated factor 2 N (TAF15) and Fused in sarcoma (FUS) are nucleic acid binding proteins belonging to the conserved FET family of proteins. They are involved in diverse processes such as pre-mRNA splicing, mRNA transport, and DNA binding. The absence of information regarding the structural mechanism employed by the FET family in recognizing and discriminating their cognate and non-cognate RNA targets has hampered the attainment of consensus on modes of protein-RNA binding for this family. Our study provides a molecular basis of this RNA recognition using a combination of solution-state NMR spectroscopy, calorimetry, docking and molecular dynamics simulation. Analysis of TAF15-RRM solution structure and its binding with stem-loop RNA has yielded conclusive evidence of a non-canonical mode of RNA recognition. Rather than classical stacking interactions that occur across nitrogen bases and aromatic amino acids on ribonucleoprotein sites, moderate-affinity hydrogen bonding network between the nitrogen bases in the stem-loop RNA and a concave face on the RRM surface primarily mediate TAF15-RRM RNA interaction. We have compared the binding affinities across a set of single-stranded RNA oligonucleotides to conclusively establish that RNA binding is dependent upon structural elements in the RNA rather than sequence.

  18. Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

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    Kim Dongku

    2006-11-01

    Full Text Available Abstract Background Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. Results While identifying putative ubiquitin specific protease (USP enzymes that contain a conserved Asp (I domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N, from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA. Conclusion Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.

  19. The expanding universe of ribonucleoproteins: of novel RNA-binding proteins and unconventional interactions.

    Science.gov (United States)

    Beckmann, Benedikt M; Castello, Alfredo; Medenbach, Jan

    2016-06-01

    Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.

  20. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  1. The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins

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    Ning Li

    2015-12-01

    Full Text Available Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

  2. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    Science.gov (United States)

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  3. Cold-inducible RNA binding protein (CIRP, a novel XTcf-3 specific target gene regulates neural development in Xenopus

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    Wedlich Doris

    2008-08-01

    Full Text Available Abstract Background As nuclear mediators of wnt/β-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/β-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants. Results We identified cold-inducible RNA binding protein (CIRP as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3. Conclusion Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.

  4. Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS.

    Science.gov (United States)

    Teplova, Marianna; Farazi, Thalia A; Tuschl, Thomas; Patel, Dinshaw J

    2016-01-01

    RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutations in vivo.

  5. Oxidative stress alters physiological and morphological neuronal properties.

    Science.gov (United States)

    Hasan, Sonia M; Joe, Mary; Alshuaib, Waleed B

    2007-07-01

    We investigated the effects of H(2)O(2)-induced oxidative stress on the delayed-rectifier current (IK(DR)), neuronal physiological and morphological properties. Measurements were obtained from hippocampal CA1 neurons in control solution and from the same neurons after exposure to oxidative stress (short- and long-term H(2)O(2) external applications at 0.1, 1, and 10 mM). With short-term (6 min) H(2)O(2) (1 mM) treatment, IK(DR) measured in the H(2)O(2)-containing solution (778 +/- 23 pA, n=20), was smaller than that measured in the control Ca(2+)-free Hepes solution (1,112 +/- 38 pA, n=20). Coenzyme Q(10) (0.1 mM) pretreatment prevented the H(2)O(2)-induced inhibition of IK(DR). With long-term (40, 80 min) H(2)O(2) (0.1, 10 mM) treatment, the neuron lost its distinctive shape (rounded up) and the neurite almost disappeared. These results suggest that oxidative stress, which inhibits IK(DR), can alter neural activity. The morphological changes caused by H(2)O(2) support the idea that oxidative stress causes intracellular damage and compromises neural function.

  6. Chromophore Deprotonation State Alters the Optical Properties of Blue Chromoprotein.

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    Cheng-Yi Chiang

    Full Text Available Chromoproteins (CPs have unique colors and can be used in biological applications. In this work, a novel blue CP with a maximum absorption peak (λmax at 608 nm was identified from the carpet anemone Stichodactyla gigantea (sgBP. In vivo expression of sgBP in zebrafish would change the appearance of the fishes to have a blue color, indicating the potential biomarker function. To enhance the color properties, the crystal structure of sgBP at 2.25 Å resolution was determined to allow structure-based protein engineering. Among the mutations conducted in the Gln-Tyr-Gly chromophore and chromophore environment, a S157C mutation shifted the λmax to 604 nm with an extinction coefficient (ε of 58,029 M-1·cm-1 and darkened the blue color expression. The S157C mutation in the sgBP chromophore environment could affect the color expression by altering the deprotonation state of the phenolic group in the chromophore. Our results provide a structural basis for the blue color enhancement of the biomarker development.

  7. DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

    Science.gov (United States)

    Dutertre, Martin; Vagner, Stéphan

    2016-09-29

    Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs.

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

    Science.gov (United States)

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

    2009-05-01

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

  9. Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing.

    Science.gov (United States)

    Tsai, Pei-Ling; Chiou, Ni-Ting; Kuss, Sharon; García-Sastre, Adolfo; Lynch, Kristen W; Fontoura, Beatriz M A

    2013-01-01

    Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.

  10. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

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    Gabriela Huelgas-Morales

    2016-04-01

    Full Text Available In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.

  11. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Dunkle, Jack A.; Wang, Leyi; Feldman, Michael B.; Pulk, Arto; Chen, Vincent B.; Kapral, Gary J.; Noeske, Jonas; Richardson, Jane S.; Blanchard, Scott C.; Cate, Jamie H. Doudna (Cornell); (UCB); (Duke)

    2011-09-06

    During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of {approx}3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

  12. A method for in vivo identification of bacterial small RNA-binding proteins.

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    Osborne, Jonathan; Djapgne, Louise; Tran, Bao Quoc; Goo, Young Ah; Oglesby-Sherrouse, Amanda G

    2014-12-01

    Small bacterial regulatory RNAs (sRNAs) have gained immense appreciation over the last decade for their roles in mediating posttranscriptional gene regulation of numerous physiological processes. Several proteins contribute to sRNA stability and regulation, most notably the Hfq RNA-binding protein. However, not all sRNAs rely on Hfq for their stability. It is therefore likely that other proteins contribute to the stability and function of certain bacterial sRNAs. Here, we describe a methodology for identifying in vivo-binding proteins of sRNAs, developed using the iron-responsive PrrF and PrrH sRNAs of Pseudomonas aeruginosa. RNA was isolated from iron-depleted cultures, which were irradiated to cross-link nucleoprotein complexes. Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins. This method identified Hfq as a potential PrrF- and PrrH-binding protein. Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo. Hfq binding to the PrrF and PrrH sRNAs was validated by electrophoretic mobility shift assays with purified Hfq protein from P. aeruginosa. As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.

  13. Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition.

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    Madina, Bhaskara R; Kumar, Vikas; Metz, Richard; Mooers, Blaine H M; Bundschuh, Ralf; Cruz-Reyes, Jorge

    2014-07-01

    Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.

  14. Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

    Science.gov (United States)

    Kharraz, Yacine; Salmand, Pierre-Adrien; Camus, Anne; Auriol, Jacques; Gueydan, Cyril; Kruys, Véronique; Morello, Dominique

    2010-01-01

    Background TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. Methodology/Principal Findings To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR) allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2α that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. Conclusions/Significance This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming. PMID:20596534

  15. Impaired embryonic development in mice overexpressing the RNA-binding protein TIAR.

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    Yacine Kharraz

    Full Text Available BACKGROUND: TIA-1-related (TIAR protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs. Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. METHODOLOGY/PRINCIPAL FINDINGS: To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2alpha that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. CONCLUSIONS/SIGNIFICANCE: This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming.

  16. In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase.

    Science.gov (United States)

    Clark, Daniel N; Jones, Scott A; Hu, Jianming

    2017-01-01

    The hepatitis B virus (HBV) polymerase synthesizes the viral DNA genome from the pre-genomic RNA (pgRNA) template through reverse transcription. Initiation of viral DNA synthesis is accomplished via a novel protein priming mechanism, so named because the polymerase itself acts as a primer, whereby the initiating nucleotide becomes covalently linked to a tyrosine residue on the viral polymerase. Protein priming, in turn, depends on specific recognition of the packaging signal on pgRNA called epsilon. These early events in viral DNA synthesis can now be dissected in vitro as described here.The polymerase is expressed in mammalian cells and purified by immunoprecipitation. The purified protein is associated with host cell factors, is enzymatically active, and its priming activity is epsilon dependent. A minimal epsilon RNA construct from pgRNA is co-expressed with the polymerase in cells. This RNA binds to and co-immunoprecipitates with the polymerase. Modifications can be made to either the epsilon RNA or the polymerase protein by manipulating the expression plasmids. Also, the priming reaction itself can be modified to assay for the initiation or subsequent DNA synthesis during protein priming, the susceptibility of the polymerase to chemical inhibitors, and the precise identification of the DNA products upon their release from the polymerase. The identity of associated host factors can also be evaluated. This protocol closely mirrors our current understanding of the RNA binding and protein priming steps of the HBV replication cycle, and it is amenable to modification. It should therefore facilitate both basic research and drug discovery.

  17. Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus.

    Science.gov (United States)

    Martínez-Turiño, Sandra; Hernández, Carmen

    2010-12-01

    Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5'-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from Escherichia coli and tested its ability to bind RNA in vitro. The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.

  18. Plakophilin-associated RNA-binding proteins in prostate cancer and their implications in tumor progression and metastasis

    NARCIS (Netherlands)

    Yang, Cheng; Stroebel, Philipp; Marx, Alexander; Hofmann, Ilse

    2013-01-01

    Both plakophilins (PKP) 1 and 3 play a role in the progression of prostate cancer. The RNA-binding proteins (RBPs) GAP-SH3-binding protein (G3BP), fragile-X-related protein 1 (FXR1), poly(A)-binding protein, cytoplasmic 1 (PABPC1), and up-frameshift factor 1 (UPF1) are associated with PKP3. All thes

  19. Anti-Japanese-encephalitis-viral effects of kaempferol and daidzin and their RNA-binding characteristics.

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    Ting Zhang

    Full Text Available BACKGROUND: New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV infections. JEV requires an α-1 translational frameshift to synthesize the NS1' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae and isoflavonoid daidzin (Dai against JEV have not been described. METHODOLOGY/PRINCIPAL FINDINGS: The 50% cytotoxic concentration (CC(50 and 50% effective concentration (EC(50 against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC(50 values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC(50 was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent K(b value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could. CONCLUSIONS/SIGNIFICANCE: Kae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA.

  20. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Ayaho; Kanaba, Teppei [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Satoh, Ryosuke [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Fujiwara, Toshinobu [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku,Nagoya 467-8603 (Japan); Ito, Yutaka [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Sugiura, Reiko [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Mishima, Masaki, E-mail: mishima-masaki@tmu.ac.jp [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan)

    2013-07-19

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.

  1. Split End Family RNA Binding Proteins: Novel Tumor Suppressors Coupling Transcriptional Regulation with RNA Processing

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    Hairui Su

    2015-01-01

    Full Text Available Split End (SPEN family proteins have three members: SPEN, RBM15, and RBM15B. SPEN family proteins contain three conserved RNA recognition motifs on the N-terminal region and an SPOC domain on the C-terminal region. RBM15 is fused to MKL1 in chromosome translocation t (1;22, which causes childhood acute megakaryoblastic leukemia (AMKL. Haploinsufficiency of RBM15 in AMKL indicates that RBM15 is a tumor suppressor. Both SPEN and RBM15 are mutated in a variety of cancer types, implying that they are tumor suppressors. SPEN and RBM15are required for the development of multiple organs including hematopoiesis partly via regulating the NOTCH signaling pathway, as well as the WNT signaling pathway in species ranging from Drosophila to mammals. Besides transcriptional regulation, RBM15 regulates RNA export and RNA splicing. In this review, we summarized data in the literature on how the members in SPEN family regulate gene expression at transcription and RNA processing steps. The crosstalk between epigenetic regulation and RNA metabolism is increasingly appreciated in understanding tumorigenesis. Studying the SPEN family of RNA binding proteins will create new perspectives for cancer therapy.

  2. Linking Spermatid Ribonucleic Acid (RNA) Binding Protein and Retrogene Diversity to Reproductive Success*

    Science.gov (United States)

    Chapman, Karen M.; Powell, Heather M.; Chaudhary, Jaideep; Shelton, John M.; Richardson, James A.; Richardson, Timothy E.; Hamra, F. Kent

    2013-01-01

    Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness. PMID:23938467

  3. ATtRACT-a database of RNA-binding proteins and associated motifs.

    Science.gov (United States)

    Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

    2016-01-01

    RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available athttp://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid-F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discoveringde novomotifs enriched in a set of related sequences and compare them with the motifs included in the database.Database URL:http:// attract. cnic. es.

  4. Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels

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    G. Nagarajan

    2014-03-01

    Full Text Available The dsRNA binding protein (RBP encoding gene of parapoxviruses (PPVs from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV and PCPV (reindeer PCPV and human PCPV shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV from different geographical areas of the world shared 69.5–71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV in comparison to caprine and ovine PPV (ORFV.

  5. RNA binding proteins in spermatogenesis: an in depth focus on the Musashi family

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    Jessie M Sutherland

    2015-01-01

    Full Text Available Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the complex process of mammalian spermatogenesis, male germ cells experience extended periods of the inactive transcription despite heavy translational requirements for continued growth and differentiation. Hence, spermatogenesis is highly reliant on mechanisms of posttranscriptional regulation of gene expression, facilitated by RNA binding proteins (RBPs, which remain abundantly expressed throughout this process. One such group of proteins is the Musashi family, previously identified as critical regulators of testis germ cell development and meiosis in Drosophila, and also shown to be vital to sperm development and reproductive potential in the mouse. This review describes the role and function of RBPs within the scope of male germ cell development, focusing on our recent knowledge of the Musashi proteins in spermatogenesis. The functional mechanisms utilized by RBPs within the cell are outlined in depth, and the significance of sub-cellular localization and stage-specific expression in relation to the mode and impact of posttranscriptional regulation is also highlighted. We emphasize the historical role of the Musashi family of RBPs in stem cell function and cell fate determination, as originally characterized in Drosophila and Xenopus, and conclude with our current understanding of the differential roles and functions of the mammalian Musashi proteins, Musashi-1 and Musashi-2, with a primary focus on our findings in spermatogenesis. This review highlights both the essential contribution of RBPs to posttranscriptional regulation and the importance of the Musashi family as master regulators of male gamete development.

  6. Conformational-Dependent and Independent RNA Binding to the Fragile X Mental Retardation Protein

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    Xin Yan

    2011-01-01

    Full Text Available The interaction between the fragile X mental retardation protein (FMRP and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP.

  7. The extended AT-hook is a novel RNA binding motif.

    Science.gov (United States)

    Filarsky, Michael; Zillner, Karina; Araya, Ingrid; Villar-Garea, Ana; Merkl, Rainer; Längst, Gernot; Németh, Attila

    2015-01-01

    The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12-15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.

  8. In vitro RNA-binding assay for studying trans-factors for RNA editing in chloroplasts.

    Science.gov (United States)

    Shikanai, Toshiharu; Okuda, Kenji

    2011-01-01

    In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially powerful with a combination of in vivo analyses based on the phenotypes of mutants and transgenic plants. In this chapter, we describe methods for the expression of recombinant PPR proteins in Escherichia coli, preparation of probe RNAs, and RNA gel shift assays. These methods can also be utilized for other RNA-binding proteins.

  9. A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity.

    Science.gov (United States)

    Stumpf, Craig R; Kimble, Judith; Wickens, Marvin

    2008-08-01

    PUF proteins comprise a highly conserved family of sequence-specific RNA binding proteins that regulate target mRNAs via binding directly to their 3'UTRs. The Caenorhabditis elegans genome encodes several PUF proteins, which cluster into four groups based on sequence similarity; all share amino acids that interact with the RNA in the cocrystal of human Pumilio with RNA. Members of the FBF and the PUF-8/9 groups bind different but related RNA sequences. We focus here on the binding specificity of representatives of a third cluster, comprising PUF-5, -6, and -7. We performed in vivo selection experiments using the yeast three-hybrid system to identify RNA sequences that bind PUF-5 and PUF-6, and we confirmed binding to optimal sites in vitro. The consensus sequences derived from the screens are similar for PUF-5 and PUF-6 but differ from those of the FBF or PUF-8/-9 groups. Similarly, neither PUF-5 nor PUF-6 bind the recognition sites preferred by the other clusters. Mutagenesis studies confirmed the unique RNA specificity of PUF-5/-6. Using the PUF-5 consensus derived from our experiments, we searched a database of C. elegans 3'UTRs to identify potential targets of PUF-5, several of which indeed bind PUF-5. Therefore the consensus has predictive value and provides a route to finding genuine targets of these proteins.

  10. A new generation of proto-oncogenes: cold-inducible RNA binding proteins.

    Science.gov (United States)

    Lleonart, M E

    2010-01-01

    This review focuses on the roles of two major cold-inducible RNA binding proteins known in human cells: CIRP and RBM3. Both proteins were discovered when they were shown to be induced after exposure to a moderate cold-shock and other cellular stresses such as UV radiation and hypoxia. Initially, it was suggested that these proteins have a suppressive rather stimulatory effect on proliferation; however, proliferative and/or proto-oncogenic functions have recently been assigned to CIRP and RBM3. In a high throughput genetic screen, we recently identified CIRP as an immortalized gene in murine primary cells. On the other hand, the role of RBM3 in transformation has already been demonstrated. Interestingly, both CIRP and RBM3 have been found to be up-regulated in human tumors. This article highlights the roles of CIRP and RBM3 in tumorigenesis, and proposes a model by which CIRP might contribute to senescence bypass by counteracting the deleterious effects of oxidative damage.

  11. DO-RIP-seq to quantify RNA binding sites transcriptome-wide.

    Science.gov (United States)

    Nicholson, Cindo O; Friedersdorf, Matthew B; Bisogno, Laura S; Keene, Jack D

    2016-11-10

    Post-transcriptional processes orchestrate gene expression through dynamic protein-RNA interactions. These interactions occur at specific sites determined by RNA sequence, secondary structure, or nucleotide modifications. Methods have been developed either to quantify binding of whole transcripts or to identify the binding sites, but there is none proven to quantify binding at both the whole transcript and binding site levels. Here we describe digestion optimized RNA immunoprecipitation with deep sequencing (DO-RIP-seq) as a method that quantitates at the whole transcript target (RIP-Seq-Like or RSL) level and at the binding site level (BSL) using continuous metrics. DO-RIP-seq methodology was developed using the RBP HuR/ELAVL1 as a test case (Nicholson et al., 2016). DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions. Analyses of sequenced cDNA libraries made from the bound RNA fragments yielded two types of enrichment scores; one for RSL binding events and the other for BSL events (Nicholson et al., 2016). These analyses plus the extensive read coverage of DO-RIP-seq allows seamless integration of binding site and whole transcript information. Therefore, DO-RIP-seq is useful for quantifying RBP binding events that are regulated during dynamic biological processes.

  12. Myofibrillar disruption and RNA-binding protein aggregation in a mouse model of limb-girdle muscular dystrophy 1D.

    Science.gov (United States)

    Bengoechea, Rocio; Pittman, Sara K; Tuck, Elizabeth P; True, Heather L; Weihl, Conrad C

    2015-12-01

    Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominantly inherited missense mutations in DNAJB6, an Hsp40 co-chaperone. LGMD1D muscle has rimmed vacuoles and inclusion bodies containing DNAJB6, Z-disc proteins and TDP-43. DNAJB6 is expressed as two isoforms; DNAJB6a and DNAJB6b. Both isoforms contain LGMD1D mutant residues and are expressed in human muscle. To identify which mutant isoform confers disease pathogenesis and generate a mouse model of LGMD1D, we evaluated DNAJB6 expression and localization in skeletal muscle as well as generating DNAJB6 isoform specific expressing transgenic mice. DNAJB6a localized to myonuclei while DNAJB6b was sarcoplasmic. LGMD1D mutations in DNAJB6a or DNAJB6b did not alter this localization in mouse muscle. Transgenic mice expressing the LGMD1D mutant, F93L, in DNAJB6b under a muscle-specific promoter became weak, had early lethality and developed muscle pathology consistent with myopathy after 2 months; whereas mice expressing the same F93L mutation in DNAJB6a or overexpressing DNAJB6a or DNAJB6b wild-type transgenes remained unaffected after 1 year. DNAJB6b localized to the Z-disc and DNAJB6b-F93L expressing mouse muscle had myofibrillar disorganization and desmin inclusions. Consistent with DNAJB6 dysfunction, keratin 8/18, a DNAJB6 client also accumulated in DNAJB6b-F93L expressing mouse muscle. The RNA-binding proteins hnRNPA1 and hnRNPA2/B1 accumulated and co-localized with DNAJB6 at sarcoplasmic stress granules suggesting that these proteins maybe novel DNAJB6b clients. Similarly, hnRNPA1 and hnRNPA2/B1 formed sarcoplasmic aggregates in patients with LGMD1D. Our data support that LGMD1D mutations in DNAJB6 disrupt its sarcoplasmic function suggesting a role for DNAJB6b in Z-disc organization and stress granule kinetics.

  13. Amphotericin B nephrotoxicity: the adverse consequences of altered membrane properties.

    Science.gov (United States)

    Sawaya, B P; Briggs, J P; Schnermann, J

    1995-08-01

    Amphotericin B (AmB) has been in clinical use for more than 30 yr but has remained the most effective drug for treatment of serious fungal infections. Its use has increased in recent years, as the result of increases in aggressive intensive care support and increased numbers of immunocompromised patients. Nephrotoxic manifestations are common, and this is the major factor limiting the clinical use of the drug. A number of recent studies have contributed to a better understanding of the mechanism by which AmB exerts its nephrotoxic effect. AmB alters cell membrane permeability and probably as a consequence alters tubular and vascular smooth muscle cell function, leading to various tubular transport defects and vasoconstriction. Decreased RBF appears to play a major role in AmB-induced reduction GFR, and recurrent ischemia may be the basis of permanent structural nephrotoxic effects. Salt loading is the only measure proven by controlled prospective study to ameliorate AmB nephrotoxicity in humans. Liposomal AmB and the formulation of an emulsion of AmB in lipid may provide a protective effect based on altering the affinity of AmB for mammalian cell membranes, while preserving high efficacy against fungal cells. However, further studies are needed to evaluate the efficacy and safety of these new AmB formulations.

  14. Salicylic acid-dependent and -independent impact of an RNA-binding protein on plant immunity.

    Science.gov (United States)

    Hackmann, Christian; Korneli, Christin; Kutyniok, Magdalene; Köster, Tino; Wiedenlübbert, Matthias; Müller, Caroline; Staiger, Dorothee

    2014-03-01

    Plants overexpressing the RNA-binding protein AtGRP7 (AtGRP7-ox plants) constitutively express the PR-1 (PATHOGENESIS-RELATED-1), PR-2 and PR-5 transcripts associated with salicylic acid (SA)-mediated immunity and show enhanced resistance against Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we investigated whether the function of AtGRP7 in plant immunity depends on SA. Endogenous SA was elevated fivefold in AtGRP7-ox plants. The elevated PR-1, PR-2 and PR-5 levels were eliminated upon expression of the salicylate hydroxylase nahG in AtGRP7-ox plants and elevated PR-1 levels were suppressed by sid (salicylic acid deficient) 2-1 that is impaired in SA biosynthesis. RNA immunoprecipitation showed that AtGRP7 does not bind the PR-1 transcript in vivo, whereas it binds PDF1.2. Constitutive or inducible AtGRP7 overexpression increases PR-1 promoter activity, indicating that AtGRP7 affects PR-1 transcription. In line with this, the effect of AtGRP7 on PR-1 is suppressed by npr (non-expressor of PR genes) 1. Whereas AtGRP7-ox plants restricted growth of Pto DC3000 compared with wild type (wt), sid2-1 AtGRP7-ox plants allowed more growth than AtGRP7-ox plants. Furthermore, we show an enhanced hypersensitive response triggered by avirulent Pto DC3000 (AvrRpt2) in AtGRP7-ox compared with wt. In sid2-1 AtGRP7-ox, an intermediate phenotype was observed. Thus, AtGRP7 has both SA-dependent and SA-independent effects on plant immunity.

  15. Ablation of the Sam68 RNA binding protein protects mice from age-related bone loss.

    Directory of Open Access Journals (Sweden)

    Stéphane Richard

    2005-12-01

    Full Text Available The Src substrate associated in mitosis of 68 kDa (Sam68 is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68-/- mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68-/- mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68-/- mice. Sam68-/- bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68+/+ and Sam68-/- littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68-/- mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68-/- mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice.

  16. Novel predicted RNA-binding domains associated with the translation machinery.

    Science.gov (United States)

    Aravind, L; Koonin, E V

    1999-03-01

    Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60-65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions.

  17. AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

    Science.gov (United States)

    Xu, Kaibiao; Yang, Lele; Zhao, Danyun; Wu, Yaoyao; Qi, Huayu

    2014-06-01

    Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells

  18. Conserved regulation of MAP kinase expression by PUF RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Myon-Hee Lee

    2007-12-01

    Full Text Available Mitogen-activated protein kinase (MAPK and PUF (for Pumilio and FBF [fem-3 binding factor] RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3' untranslated region (3' UTR and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3'UTR elements in both Erk2 and p38alpha mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38alpha 3' UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP, to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.

  19. IGF2 mRNA-binding protein 2: biological function and putative role in type 2 diabetes

    DEFF Research Database (Denmark)

    Christiansen, J.; Kolte, A.M.; Hansen, T.O.;

    2009-01-01

    Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization......, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2...... and T2D Udgivelsesdato: 2009/11...

  20. Altered cross-bridge properties in skeletal muscle dystrophies

    Science.gov (United States)

    Guellich, Aziz; Negroni, Elisa; Decostre, Valérie; Demoule, Alexandre; Coirault, Catherine

    2014-01-01

    Force and motion generated by skeletal muscle ultimately depends on the cyclical interaction of actin with myosin. This mechanical process is regulated by intracellular Ca2+ through the thin filament-associated regulatory proteins i.e.; troponins and tropomyosin. Muscular dystrophies are a group of heterogeneous genetic affections characterized by progressive degeneration and weakness of the skeletal muscle as a consequence of loss of muscle tissue which directly reduces the number of potential myosin cross-bridges involved in force production. Mutations in genes responsible for skeletal muscle dystrophies (MDs) have been shown to modify the function of contractile proteins and cross-bridge interactions. Altered gene expression or RNA splicing or post-translational modifications of contractile proteins such as those related to oxidative stress, may affect cross-bridge function by modifying key proteins of the excitation-contraction coupling. Micro-architectural change in myofilament is another mechanism of altered cross-bridge performance. In this review, we provide an overview about changes in cross-bridge performance in skeletal MDs and discuss their ultimate impacts on striated muscle function. PMID:25352808

  1. Altered cross-bridge properties in skeletal muscle dystrophies.

    Science.gov (United States)

    Guellich, Aziz; Negroni, Elisa; Decostre, Valérie; Demoule, Alexandre; Coirault, Catherine

    2014-01-01

    Force and motion generated by skeletal muscle ultimately depends on the cyclical interaction of actin with myosin. This mechanical process is regulated by intracellular Ca(2+) through the thin filament-associated regulatory proteins i.e.; troponins and tropomyosin. Muscular dystrophies are a group of heterogeneous genetic affections characterized by progressive degeneration and weakness of the skeletal muscle as a consequence of loss of muscle tissue which directly reduces the number of potential myosin cross-bridges involved in force production. Mutations in genes responsible for skeletal muscle dystrophies (MDs) have been shown to modify the function of contractile proteins and cross-bridge interactions. Altered gene expression or RNA splicing or post-translational modifications of contractile proteins such as those related to oxidative stress, may affect cross-bridge function by modifying key proteins of the excitation-contraction coupling. Micro-architectural change in myofilament is another mechanism of altered cross-bridge performance. In this review, we provide an overview about changes in cross-bridge performance in skeletal MDs and discuss their ultimate impacts on striated muscle function.

  2. Altered cross-bridge properties in skeletal muscle dystrophies

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    Aziz eGuellich

    2014-10-01

    Full Text Available Force and motion generated by skeletal muscle ultimately depends on the cyclical interaction of actin with myosin. This mechanical process is regulated by intracellular Ca2+ through the thin filament-associated regulatory proteins i.e.; troponins and tropomyosin. Muscular dystrophies are a group of heterogeneous genetic affections characterized by progressive degeneration and weakness of the skeletal muscle as a consequence of loss of muscle tissue which directly reduces the number of potential myosin cross-bridges involved in force production. Mutations in genes responsible for skeletal muscle dystrophies have been shown to modify the function of contractile proteins and cross-bridge interactions. Altered gene expression or RNA splicing or post-translational modifications of contractile proteins such as those related to oxidative stress, may affect cross-bridge function by modifying key proteins of the excitation-contraction coupling. Micro-architectural change in myofilament is another mechanism of altered cross-bridge performance. In this review, we provide an overview about changes in cross-bridge performance in skeletal muscle dystrophies and discuss their ultimate impacts on striated muscle function.

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

    Science.gov (United States)

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

    2017-01-01

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

  4. THUMP--a predicted RNA-binding domain shared by 4-thiouridine, pseudouridine synthases and RNA methylases.

    Science.gov (United States)

    Aravind, L; Koonin, E V

    2001-04-01

    Sequence profile searches were used to identify an ancient domain in ThiI-like thiouridine synthases, conserved RNA methylases, archaeal pseudouridine synthases and several uncharacterized proteins. We predict that this domain is an RNA-binding domain that adopts an alpha/beta fold similar to that found in the C-terminal domain of translation initiation factor 3 and ribosomal protein S8.

  5. Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding.

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    Bogdan Tarus

    Full Text Available The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP. X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73-90, loop 2 (200-214. In NP, loops 1 and 2 formed a 10-15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91-112 that interacted with the RNA grove (linker 360-373 via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s of the nucleoprotein.

  6. Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR▿ †

    OpenAIRE

    Kim, Henry S.; Kuwano, Yuki; Zhan, Ming; Pullmann, Rudolf; Mazan-Mamczarz, Krystyna; Li, Huai; Kedersha, Nancy; Anderson, Paul; Wilce, Matthew C J; Gorospe, Myriam; Wilce, Jacqueline A.

    2007-01-01

    The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3′ untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, an...

  7. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

    2015-11-15

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  8. Clusters of basic amino acids contribute to RNA binding and nucleolar localization of ribosomal protein L22.

    Directory of Open Access Journals (Sweden)

    Jennifer L Houmani

    Full Text Available The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80-93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80-93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.

  9. Elevation alters ecosystem properties across temperate treelines globally

    Science.gov (United States)

    Mayor, Jordan R.; Sanders, Nathan J.; Classen, Aimée T.; Bardgett, Richard D.; Clément, Jean-Christophe; Fajardo, Alex; Lavorel, Sandra; Sundqvist, Maja K.; Bahn, Michael; Chisholm, Chelsea; Cieraad, Ellen; Gedalof, Ze’Ev; Grigulis, Karl; Kudo, Gaku; Oberski, Daniel L.; Wardle, David A.

    2017-01-01

    Temperature is a primary driver of the distribution of biodiversity as well as of ecosystem boundaries. Declining temperature with increasing elevation in montane systems has long been recognized as a major factor shaping plant community biodiversity, metabolic processes, and ecosystem dynamics. Elevational gradients, as thermoclines, also enable prediction of long-term ecological responses to climate warming. One of the most striking manifestations of increasing elevation is the abrupt transitions from forest to treeless alpine tundra. However, whether there are globally consistent above- and belowground responses to these transitions remains an open question. To disentangle the direct and indirect effects of temperature on ecosystem properties, here we evaluate replicate treeline ecotones in seven temperate regions of the world. We find that declining temperatures with increasing elevation did not affect tree leaf nutrient concentrations, but did reduce ground-layer community-weighted plant nitrogen, leading to the strong stoichiometric convergence of ground-layer plant community nitrogen to phosphorus ratios across all regions. Further, elevation-driven changes in plant nutrients were associated with changes in soil organic matter content and quality (carbon to nitrogen ratios) and microbial properties. Combined, our identification of direct and indirect temperature controls over plant communities and soil properties in seven contrasting regions suggests that future warming may disrupt the functional properties of montane ecosystems, particularly where plant community reorganization outpaces treeline advance.

  10. Ocean acidification alters the material properties of Mytilus edulis shells.

    Science.gov (United States)

    Fitzer, Susan C; Zhu, Wenzhong; Tanner, K Elizabeth; Phoenix, Vernon R; Kamenos, Nicholas A; Cusack, Maggie

    2015-02-01

    Ocean acidification (OA) and the resultant changing carbonate saturation states is threatening the formation of calcium carbonate shells and exoskeletons of marine organisms. The production of biominerals in such organisms relies on the availability of carbonate and the ability of the organism to biomineralize in changing environments. To understand how biomineralizers will respond to OA the common blue mussel, Mytilus edulis, was cultured at projected levels of pCO2 (380, 550, 750, 1000 µatm) and increased temperatures (ambient, ambient plus 2°C). Nanoindentation (a single mussel shell) and microhardness testing were used to assess the material properties of the shells. Young's modulus (E), hardness (H) and toughness (KIC) were measured in mussel shells grown in multiple stressor conditions. OA caused mussels to produce shell calcite that is stiffer (higher modulus of elasticity) and harder than shells grown in control conditions. The outer shell (calcite) is more brittle in OA conditions while the inner shell (aragonite) is softer and less stiff in shells grown under OA conditions. Combining increasing ocean pCO2 and temperatures as projected for future global ocean appears to reduce the impact of increasing pCO2 on the material properties of the mussel shell. OA may cause changes in shell material properties that could prove problematic under predation scenarios for the mussels; however, this may be partially mitigated by increasing temperature.

  11. Alteration of Skin Properties with Autologous Dermal Fibroblasts

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    Rajesh L. Thangapazham

    2014-05-01

    Full Text Available Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices.

  12. Differential expression of the RNA-binding motif protein 3 in human astrocytoma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-tao; ZHANG Zhi-wen; XUE Jing-hui; KONG Hai-bo; LIU Ai-jun; LI Shou-chun; LIU Yu-xiao

    2013-01-01

    Background The RNA-binding motif protein 3 (RBM3),which is transcriptionally induced by low temperature and hypoxia,has recently been found to be upregulated in human tumors.However,its expression status in human astrocytoma is not well defined.This article focuses on the differential expression of RBM3 in human astrocytomas of different grades and normal brain tissues.Methods RBM3 was detected in astrocytomas and normal brain tissues by quantitative real-time PCR,immunohistochemistry,and Western blotting.Analysis of variance was performed on the data from quantitative real-time PCR.The Fisher's exact test was used to analyze the immunohistochemistry results.A P-value of less than 0.05 indicates a statistically significant difference.Results On one hand,the mRNA expression levels of three X-chromosome-related RBM genes (RBMX,RBM3,and RBM10) were detected by quantitative real-time PCR.The results showed that there were no significant differences in RBMX and RBM10 mRNA expression levels in human astrocytomas of different grades and normal brain tissues.However,RBM3 mRNA expression levels were elevated in high-grade (World Health Organization (WHO) Grade Ⅲ-Ⅳ) astrocytomas versus low-grade (WHO Grade Ⅰ-Ⅱ) astrocytomas (5.06±0.66 vs.1.60±0.58; P <0.05) or normal controls (5.06±0.66 vs.1.03±0.22; P <0.05) as determined by quantitative real-time PCR analysis.On the other hand,immunohistochemistry showed an increased RBM3 labeling index in astrocytomas of different grades and normal brain tissues (positive staining rate:astrocytoma Grade Ⅳ,92.9%; astrocytoma Grade Ⅲ,81.8%; astrocytoma Grade Ⅰ-Ⅱ,50%;normal brain tissues,37.5%; high-grade astrocytoma versus normal brain tissues,P <0.05; high-grade astrocytoma versus low-grade astrocytoma,P <0.05).The higher protein levels of RBM3 were also validated in high-grade astrocytomas and low-grade astrocytomas compared with normal brain tissues by Western blotting.Conclusions These

  13. Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties

    Science.gov (United States)

    Orihuela, C. J.; Janssen, R.; Robb, C. W.; Watson, D. A.; Niesel, D. W.

    2000-01-01

    We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

  14. The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

    Science.gov (United States)

    Collins, Mahlon; Riascos, David; Kovalik, Tina; An, Jiyan; Krupa, Kelly; Krupa, Kristin; Hood, Brian L; Conrads, Thomas P; Renton, Alan E; Traynor, Bryan J; Bowser, Robert

    2012-11-01

    RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

  15. Altering properties of cerium oxide thin films by Rh doping

    Energy Technology Data Exchange (ETDEWEB)

    Ševčíková, Klára, E-mail: klarak.sevcikova@seznam.cz [Faculty of Mathematics and Physics, Department of Surface and Plasma Science, Charles University in Prague, V Holešovičkách 2, 180 00 Prague 8 (Czech Republic); NIMS Beamline Station at SPring-8, National Institute for Materials Science, Sayo, Hyogo 679-5148 (Japan); Nehasil, Václav, E-mail: nehasil@mbox.troja.mff.cuni.cz [Faculty of Mathematics and Physics, Department of Surface and Plasma Science, Charles University in Prague, V Holešovičkách 2, 180 00 Prague 8 (Czech Republic); Vorokhta, Mykhailo, E-mail: vorohtam@gmail.com [Faculty of Mathematics and Physics, Department of Surface and Plasma Science, Charles University in Prague, V Holešovičkách 2, 180 00 Prague 8 (Czech Republic); Haviar, Stanislav, E-mail: stanislav.haviar@gmail.com [Faculty of Mathematics and Physics, Department of Surface and Plasma Science, Charles University in Prague, V Holešovičkách 2, 180 00 Prague 8 (Czech Republic); Matolín, Vladimír, E-mail: matolin@mbox.troja.mff.cuni.cz [Faculty of Mathematics and Physics, Department of Surface and Plasma Science, Charles University in Prague, V Holešovičkách 2, 180 00 Prague 8 (Czech Republic); and others

    2015-07-15

    Highlights: • Thin films of ceria doped by rhodium deposited by RF magnetron sputtering. • Concentration of rhodium has great impact on properties of Rh–CeO{sub x} thin films. • Intensive oxygen migration in films with low concentration of rhodium. • Oxygen migration suppressed in films with high amount of Rh dopants. - Abstract: Ceria containing highly dispersed ions of rhodium is a promising material for catalytic applications. The Rh–CeO{sub x} thin films with different concentrations of rhodium were deposited by RF magnetron sputtering and were studied by soft and hard X-ray photoelectron spectroscopies, Temperature programmed reaction and X-ray powder diffraction techniques. The sputtered films consist of rhodium–cerium mixed oxide where cerium exhibits a mixed valency of Ce{sup 4+} and Ce{sup 3+} and rhodium occurs in two oxidation states, Rh{sup 3+} and Rh{sup n+}. We show that the concentration of rhodium has a great influence on the chemical composition, structure and reducibility of the Rh–CeO{sub x} thin films. The films with low concentrations of rhodium are polycrystalline, while the films with higher amount of Rh dopants are amorphous. The morphology of the films strongly influences the mobility of oxygen in the material. Therefore, varying the concentration of rhodium in Rh–CeO{sub x} thin films leads to preparing materials with different properties.

  16. Resveratrol: preventing properties against vascular alterations and ageing.

    Science.gov (United States)

    Delmas, Dominique; Jannin, Brigitte; Latruffe, Norbert

    2005-05-01

    Cardiovascular diseases are the leading cause of death in developed countries where the common pathological substrate underlying this process is atherosclerosis. Several new concepts have emerged in relation to mechanisms that contribute to the regulation of the vascular diseases and associated inflammatory effects. Recently, potential antioxidants (vitamin E, polyphenols) have received much attention as potential anti-atherosclerotic agents. Among the polyphenols with health benefic properties, resveratrol, a phytoalexin of grape, seem to be a good candidate protecting the vascular walls from oxidation, inflammation, platelet aggregation, and thrombus formation. In this review, we focus on the mechanism of resveratrol cardiovascular benefic effects. We analyze, in relation with the different steps of atherosclerotic process, the resveratrol properties at multiple levels, such as cellular signaling, enzymatic pathways, apoptosis, and gene expression. We show and discuss the relationship with reactive oxygen species, regulation of pro-inflammatory genes including cycloxygenases and cytokines in molecular inflammatory and aging processes, and how the regulation of these activites by resveratrol can lead to a prevention of vascular diseases.

  17. Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.

    Science.gov (United States)

    Farazi, Thalia A; Leonhardt, Carl S; Mukherjee, Neelanjan; Mihailovic, Aleksandra; Li, Song; Max, Klaas E A; Meyer, Cindy; Yamaji, Masashi; Cekan, Pavol; Jacobs, Nicholas C; Gerstberger, Stefanie; Bognanni, Claudia; Larsson, Erik; Ohler, Uwe; Tuschl, Thomas

    2014-07-01

    Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

  18. Role and properties of the gel formed during nuclear glass alteration: importance of gel formation conditions

    Science.gov (United States)

    Gin, S.; Ribet, I.; Couillard, M.

    2001-09-01

    A French SON 68 nuclear glass sample was experimentally altered to assess the mechanisms limiting the glass alteration kinetics, especially during the transition phase between the initial rate r0 and the final rate under silicon saturation conditions. A glass specimen was altered at the initial rate for one week to form a silicon-depleted non-protective gel; the specimen was then leached under static conditions at a glass-surface-to-solution-volume ( S/ V) ratio of 500 m-1 and the alteration kinetics were compared with those of a pristine glass specimen altered under the same conditions. Unexpectedly, after static leaching the previously leached glass was 2.7 times as altered as the pristine specimen, and the steady-state silicon concentration was twice as high for the previously leached specimen. STEM characterization of the alteration films showed that the initial non-protective gel constituted a silicon pump with respect to the glass, and that the glass alteration kinetics were limited only when a fraction of the gel became saturated with silicon, and exhibited protective properties. This work also shows that silicon recondensation was uniform at micrometer scale: the silicon hydrolyzed at the reaction interface then diffused before recondensing over a length comparable to the gel thickness. In addition to these findings, this investigation suggests a reinterpretation of the effect of the S/ V ratio on the glass alteration kinetics and on the steady-state dissolved silicon concentration.

  19. Trp RNA-binding attenuation protein: modifying symmetry and stability of a circular oligomer.

    Directory of Open Access Journals (Sweden)

    Oliver W Bayfield

    Full Text Available BACKGROUND: Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins. METHODOLOGY/PRINCIPAL FINDINGS: Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts--have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP. CONCLUSIONS/SIGNIFICANCE: We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.

  20. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

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    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  1. Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7

    Directory of Open Access Journals (Sweden)

    Hennig Lars

    2010-10-01

    Full Text Available Abstract Background The clock-controlled RNA-binding protein AtGRP7 influences circadian oscillations of its own transcript at the post-transcriptional level. To identify additional targets that are regulated by AtGRP7, transcript profiles of transgenic plants constitutively overexpressing AtGRP7 (AtGRP7-ox and wild type plants were compared. Results Approximately 1.4% of the transcripts represented on the Affymetrix ATH1 microarray showed changes in steady-state abundance upon AtGRP7 overexpression. One third of the differentially expressed genes are controlled by the circadian clock, and they show a distinct bias of their phase: The up-regulated genes preferentially peak around dawn, roughly opposite to the AtGRP7 peak abundance whereas the down-regulated genes preferentially peak at the end of the day. Further, transcripts responsive to abiotic and biotic stimuli were enriched among AtGRP7 targets. Transcripts encoding the pathogenesis-related PR1 and PR2 proteins were elevated in AtGRP7-ox plants but not in plants overexpressing AtGRP7 with a point mutation in the RNA-binding domain, indicating that the regulation involves RNA binding activity of AtGRP7. Gene set enrichment analysis uncovered components involved in ribosome function and RNA metabolism among groups of genes upregulated in AtGRP7-ox plants, consistent with its role in post-transcriptional regulation. Conclusion Apart from regulating a suite of circadian transcripts in a time-of-day dependent manner AtGRP7, both directly and indirectly, affects other transcripts including transcripts responsive to abiotic and biotic stimuli. This suggests a regulatory role of AtGRP7 in the output of the endogenous clock and a complex network of transcripts responsive to external stimuli downstream of the AtGRP7 autoregulatory circuit.

  2. RNA-binding Domain of the Key Structural Protein P7 for the Rice dwarf virus Particle Assembly

    Institute of Scientific and Technical Information of China (English)

    Bo-Xiong ZHONG; Yan-Wei SHEN; Toshihiro OMURA

    2005-01-01

    The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonas fragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method,respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonasfragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis,besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.

  3. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.

    Directory of Open Access Journals (Sweden)

    André P Gerber

    2004-03-01

    Full Text Available Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40-220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3'-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA-protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.

  4. Evolutionarily dynamic roles of a PUF RNA-binding protein in the somatic development of Caenorhabditis briggsae.

    Science.gov (United States)

    Liu, Qinwen; Haag, Eric S

    2014-05-01

    Gene duplication and divergence has emerged as an important aspect of developmental evolution. The genomes of Caenorhabditis nematodes encode an ancient family of PUF RNA-binding proteins. Most have been implicated in germline development, and are often redundant with paralogs of the same sub-family. An exception is Cbr-puf-2 (one of three Caenorhabditis briggsae PUF-2 sub-family paralogs), which is required for development past the second larval stage. Here, we provide a detailed functional characterization of Cbr-puf-2. The larval arrest of Cbr-puf-2 mutant animals is caused by inefficient breakdown of bacterial food, which leads to starvation. Cbr-puf-2 is required for the normal grinding cycle of the muscular terminal bulb during early larval stages, and is transiently expressed in this tissue. In addition, rescue of larval arrest reveals that Cbr-puf-2 also promotes normal vulval development. It is expressed in the anchor cell (which induces vulval fate) and vulval muscles, but not in the vulva precursor cells (VPCs) themselves. This contrasts with the VPC-autonomous repression of vulval development described for the Caenorhabditis elegans homologs fbf-1/2. These different roles for PUF proteins occur even as the vulva and pharynx maintain highly conserved anatomies across Caenorhabditis, indicating pervasive developmental system drift (DSD). Because Cbr-PUF-2 shares RNA-binding specificity with its paralogs and with C. elegans FBF, we suggest that functional novelty of RNA-binding proteins evolves through changes in the site of their expression, perhaps in concert with cis-regulatory evolution in target mRNAs.

  5. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  6. The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora

    OpenAIRE

    Veronica Ancona; Jae Hoon Lee; Youfu Zhao

    2016-01-01

    The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran...

  7. Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates csrB Transcription in Escherichia coli

    OpenAIRE

    Gudapaty, Seshagirirao; Suzuki, Kazushi; Wang, Xin; Babitzke, Paul; Romeo, Tony

    2002-01-01

    The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, f...

  8. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR.

    OpenAIRE

    Beck, A.R.; Medley, Q G; O'Brien, S; Anderson, P; Streuli, M

    1996-01-01

    TIA-1 and TIAR are RNA binding proteins of the RNA recognition motif (RRM)/ribonucleoprotein (RNP) family that have been implicated as effectors of apoptotic cell death. We report the structures of murine TIA-1 and TIAR (mTIA-1 and mTIAR) deduced from cDNA cloning, the mRNA and protein tissue distribution of mTIA-1 and mTIAR, and the exon-intron structures of the mTIA-1 and mTIAR genes. Both mTIA-1 and mTIAR are comprised of three approximately 100 amino acid N-terminal RRM domains and a appr...

  9. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development

    DEFF Research Database (Denmark)

    Nielsen, J; Christiansen, J; Lykke-Andersen, J;

    1999-01-01

    Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins.......5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development....

  10. Altered multiaxial mechanical properties of the porcine anterior lens capsule cultured in high glucose.

    Science.gov (United States)

    Pedrigi, R M; Staff, E; David, G; Glenn, S; Humphrey, J D

    2007-02-01

    Hyperglycemia can alter the mechanical properties of tissues through the formation of advanced glycation endproducts in matrix proteins that have long half-lives. We used a custom experimental system and subdomain finite element method to quantify alterations in the regional multiaxial mechanical properties of porcine lens capsules that were cultured for 8 or 14 weeks in high glucose versus control media. Findings revealed that high glucose significantly stiffened the capsules in both the circumferential and the meridional directions, but it did not affect the known regional variations in anisotropy. Such information could be important in the design of both improved clinical procedures and intraocular implants for diabetic patients.

  11. The Human dsRNA binding protein PACT is unable to functionally substitute for the Drosophila dsRNA binding protein R2D2 [v1; ref status: indexed, http://f1000r.es/201

    Directory of Open Access Journals (Sweden)

    Benjamin K Dickerman

    2013-10-01

    Full Text Available The primary function of the dsRNA binding protein (dsRBP PACT/RAX is to activate the dsRNA dependent protein kinase PKR in response to stress signals.  Additionally, it has been identified as a component of the small RNA processing pathway.  A role for PACT/RAX in this pathway represents an important interplay between two modes of post-transcriptional gene regulation.  The function of PACT/RAX in this context is poorly understood.  Thus, additional models are required to clarify the mechanism by which PACT/RAX functions.  In this study, Drosophila melanogaster was employed to identify functionally orthologous dsRNA-binding proteins.  Transgenic Drosophila expressing human PACT were generated to determine whether PACT is capable of functionally substituting for the Drosophila dsRBP R2D2, which has a well-defined role in small RNA biogenesis.  Results presented here indicate that PACT is unable to substitute for R2D2 at the whole organism level.

  12. PiRaNhA: a server for the computational prediction of RNA-binding residues in protein sequences

    Science.gov (United States)

    Murakami, Yoichi; Spriggs, Ruth V.; Nakamura, Haruki; Jones, Susan

    2010-01-01

    The PiRaNhA web server is a publicly available online resource that automatically predicts the location of RNA-binding residues (RBRs) in protein sequences. The goal of functional annotation of sequences in the field of RNA binding is to provide predictions of high accuracy that require only small numbers of targeted mutations for verification. The PiRaNhA server uses a support vector machine (SVM), with position-specific scoring matrices, residue interface propensity, predicted residue accessibility and residue hydrophobicity as features. The server allows the submission of up to 10 protein sequences, and the predictions for each sequence are provided on a web page and via email. The prediction results are provided in sequence format with predicted RBRs highlighted, in text format with the SVM threshold score indicated and as a graph which enables users to quickly identify those residues above any specific SVM threshold. The graph effectively enables the increase or decrease of the false positive rate. When tested on a non-redundant data set of 42 protein sequences not used in training, the PiRaNhA server achieved an accuracy of 85%, specificity of 90% and a Matthews correlation coefficient of 0.41 and outperformed other publicly available servers. The PiRaNhA prediction server is freely available at http://www.bioinformatics.sussex.ac.uk/PIRANHA. PMID:20507911

  13. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

    Science.gov (United States)

    Lee, Nara; Yario, Therese A; Gao, Jessica S; Steitz, Joan A

    2016-03-22

    Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.

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

    Science.gov (United States)

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

    2016-05-01

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

  15. RNAcontext: a new method for learning the sequence and structure binding preferences of RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Hilal Kazan

    Full Text Available Metazoan genomes encode hundreds of RNA-binding proteins (RBPs. These proteins regulate post-transcriptional gene expression and have critical roles in numerous cellular processes including mRNA splicing, export, stability and translation. Despite their ubiquity and importance, the binding preferences for most RBPs are not well characterized. In vitro and in vivo studies, using affinity selection-based approaches, have successfully identified RNA sequence associated with specific RBPs; however, it is difficult to infer RBP sequence and structural preferences without specifically designed motif finding methods. In this study, we introduce a new motif-finding method, RNAcontext, designed to elucidate RBP-specific sequence and structural preferences with greater accuracy than existing approaches. We evaluated RNAcontext on recently published in vitro and in vivo RNA affinity selected data and demonstrate that RNAcontext identifies known binding preferences for several control proteins including HuR, PTB, and Vts1p and predicts new RNA structure preferences for SF2/ASF, RBM4, FUSIP1 and SLM2. The predicted preferences for SF2/ASF are consistent with its recently reported in vivo binding sites. RNAcontext is an accurate and efficient motif finding method ideally suited for using large-scale RNA-binding affinity datasets to determine the relative binding preferences of RBPs for a wide range of RNA sequences and structures.

  16. Evaluation the susceptibility of five polymorphisms in microRNA-binding sites to female breast cancer risk in Chinese population.

    Science.gov (United States)

    He, Bang-Shun; Pan, Yu-Qin; Lin, Kang; Ying, Hou-Qun; Wang, Feng; Deng, Qi-Wen; Sun, Hui-Ling; Gao, Tian-Yi; Wang, Shu-Kui

    2015-11-15

    Polymorphisms in microRNA (miRNA) binding site have been widely discussed to be associated with cancer risk; however, the associations were unclear in Chinese population. To investigate the associations of five polymorphisms (rs11097457, rs1434536, rs1970801, rs1044129, rs11169571) in miRNA binding sites with breast cancer risk, a total of 435 female patients with breast cancer and 439 age- and gender-matched tumor-free individuals were enrolled in this case-control study. Sequenom MassARRAY was applied to detect the polymorphisms, and the immunohistochemistry assay was used to measure the expression of estrogen receptor (ER) and progesterone receptor (PR) and CerbB-2. The data showed that these polymorphisms were not associated with breast cancer risk or clinical characters of breast cancer in all participants and sub-group with the exception that, in the sub-group of women with their first menstruation after 14 years old, those who carried rs1970801 T allele (genotype TT/GT) were associated with decreased breast cancer risk. In short, this case-control study provided the evidence that women with their first menstruation after 14 years old and carried rs1970801 T allele (genotype TT/GT) were associated with decreased breast cancer risk.

  17. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic.

    Science.gov (United States)

    Lee, Youn-Bok; Chen, Han-Jou; Peres, João N; Gomez-Deza, Jorge; Attig, Jan; Stalekar, Maja; Troakes, Claire; Nishimura, Agnes L; Scotter, Emma L; Vance, Caroline; Adachi, Yoshitsugu; Sardone, Valentina; Miller, Jack W; Smith, Bradley N; Gallo, Jean-Marc; Ule, Jernej; Hirth, Frank; Rogelj, Boris; Houart, Corinne; Shaw, Christopher E

    2013-12-12

    The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

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

    KAUST Repository

    Gomes-Santos, Carina S. S.

    2011-05-19

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

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

    Directory of Open Access Journals (Sweden)

    Carina S S Gomes-Santos

    2011-05-01

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

  20. The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite.

    Directory of Open Access Journals (Sweden)

    Katja Müller

    Full Text Available Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2⁻ sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.

  1. Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins

    Science.gov (United States)

    Schiera, Gabriella; Di Liegro, Carlo Maria; Puleo, Veronica; Colletta, Oriana; Fricano, Anna; Cancemi, Patrizia; Di Cara, Gianluca; Di Liegro, Italia

    2016-01-01

    Extracellular vesicles (EVs) are now recognized as a fundamental way for cell-to-cell horizontal transfer of properties, in both physiological and pathological conditions. Most of EV-mediated cross-talk among cells depend on the exchange of proteins, and nucleic acids, among which mRNAs, and non-coding RNAs such as different species of miRNAs. Cancer cells, in particular, use EVs to discard molecules which could be dangerous to them (for example differentiation-inducing proteins such as histone H1.0, or antitumor drugs), to transfer molecules which, after entering the surrounding cells, are able to transform their phenotype, and even to secrete factors, which allow escaping from immune surveillance. Herein we report that melanoma cells not only secrete EVs which contain a modified form of H1.0 histone, but also transport the corresponding mRNA. Given the already known role in tumorigenesis of some RNA binding proteins (RBPs), we also searched for proteins of this class in EVs. This study revealed the presence in A375 melanoma cells of at least three RBPs, with apparent MW of about 65, 45 and 38 kDa, which are able to bind H1.0 mRNA. Moreover, we purified one of these proteins, which by MALDI-TOF mass spectrometry was identified as the already known transcription factor MYEF2. PMID:27633859

  2. α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

    Science.gov (United States)

    Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

    2014-01-01

    Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

  3. RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides

    Directory of Open Access Journals (Sweden)

    Charles R. Cantor

    2011-03-01

    Full Text Available Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation [1-3]. The RNA is tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development [1-3]. Here we describe a new version of the RNA labeling method where fluorescent protein complementation is triggered by paired interactions of two different closely-positioned RNA aptamers with two different RNA-binding viral peptides. The new method, which has been developed in bacteria as a model system, uses a smaller ribonucleoprotein complementation complex, as compared with the method using split RNA-binding protein, and it can potentially be applied to a broad variety of RNA targets in both prokaryotic and eukaryotic cells. We also describe experiments exploring background fluorescence in these RNA detection systems and conditions that improve the signal-to-background ratio.

  4. MicroRNA binding site polymorphisms as biomarkers in cancer management and research

    OpenAIRE

    Cipollini M; Landi S; Gemignani F

    2014-01-01

    Monica Cipollini, Stefano Landi*, Federica Gemignani* Department of Biology, University of Pisa, Pisa, Italy *These authors contributed equally to this work Abstract: MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression. They have been implicated in a broad range of biological processes, and miRNA-related genetic alterations probably underlie several human diseases. Single nucleotide polymorphisms of transcripts may modulate the posttranscriptional regulation of gene e...

  5. Altered cell wall properties are responsible for ammonium-reduced aluminium accumulation in rice roots.

    Science.gov (United States)

    Wang, Wei; Zhao, Xue Qiang; Chen, Rong Fu; Dong, Xiao Ying; Lan, Ping; Ma, Jian Feng; Shen, Ren Fang

    2015-07-01

    The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH4(+)) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4(+) nor NO3(-) altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4(+)-supplied roots displayed lower Al-binding capacity than those from NO3(-)-supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3(-)-supplied roots, NH4(+)-supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4(+)-reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4(+) uptake rather than direct competition for the cell wall binding sites between Al(3+) and NH4(+).

  6. The Arabidopsis KH-Domain RNA-Binding Protein ESR1 Functions in Components of Jasmonate Signalling, Unlinking Growth Restraint and Resistance to Stress.

    Directory of Open Access Journals (Sweden)

    Louise F Thatcher

    Full Text Available Glutathione S-transferases (GSTs play important roles in the protection of cells against toxins and oxidative damage where one Arabidopsis member, GSTF8, has become a commonly used marker gene for early stress and defense responses. A GSTF8 promoter fragment fused to the luciferase reporter gene was used in a forward genetic screen for Arabidopsis mutants with up-regulated GSTF8 promoter activity. This identified the esr1-1 (enhanced stress response 1 mutant which also conferred increased resistance to the fungal pathogen Fusarium oxysporum. Through positional cloning, the ESR1 gene was found to encode a KH-domain containing RNA-binding protein (At5g53060. Whole transcriptome sequencing of esr1-1 identified altered expression of genes involved in responses to biotic and abiotic stimuli, hormone signaling pathways and developmental processes. In particular was an overall significant enrichment for jasmonic acid (JA mediated processes in the esr1-1 down-regulated dataset. A subset of these genes were tested for MeJA inducibility and we found the expression of some but not all were reduced in esr1-1. The esr1-1 mutant was not impaired in other aspects of JA-signalling such as JA- sensitivity or development, suggesting ESR1 functions in specific components of the JA-signaling pathway. Examination of salicylic acid (SA regulated marker genes in esr1-1 showed no increase in basal or SA induced expression suggesting repression of JA-regulated genes is not due to antagonistic SA-JA crosstalk. These results define new roles for KH-domain containing proteins with ESR1 unlinking JA-mediated growth and defense responses.

  7. Integrative approach detected association between genetic variants of microRNA binding sites of TLRs pathway genes and OSCC susceptibility in Chinese Han population.

    Directory of Open Access Journals (Sweden)

    Yun Wang

    Full Text Available Oral squamous cell carcinoma (OSCC is a leading malignancy worldwide; the overall 5-year survival rate is approximately 50%. A variety of proteins in Toll-like receptors (TLRs pathway have been related with the risk of OSCC. However, the influence of genetic variations in TLRs pathway genes on OSCC susceptibility is unclear. Previous studies mainly focused on the coding region of genes, while the UTR region remains unstudied. In the current study, a bioinformatics approach was performed to select candidate single nucleotide polymorphisms (SNPs on microRNA binding sites of TLRs pathway genes related with OSCC. After screening 90 OSCC related TLRs pathway genes, 16 SNPs were selected for genotyping. We found that rs5030486, the polymorphisms on 3' UTR of TRAF6, was significantly associated with OSCC risk. AG genotype of TRAF6 was strongly associated with a decreased risk of OSCC (OR = 0.252; 95% CI = 0.106, 0.598; p = 0.001. In addition, AG genotype was also related with a reduced risk of OSCC progression both in univariable analysis (HR = 0.303, 95% CI = 0.092, 0.995 and multivariable analysis (HR = 0.272, 95% CI = 0.082, 0.903. Furthermore, after detecting the mRNA expression level of TRAF6 in 24 OSCC patients, we found that TRAF6 expression level was significantly different between patients carrying different genotypes at locus rs5030486 (p = 0.013, indicating that rs5030486 of TRAF6 might contribute to OSCC risk by altering TRAF6 expression level. In general, these data indicated that SNP rs5030486 could be a potential bio-marker for OSCC risk and our results might provide new insights into the association of polymorphisms within the non-coding area of genes with cancers.

  8. The Role of RNA Binding Proteins in Insulin Messenger Stability and Translation

    OpenAIRE

    2010-01-01

    Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and trans...

  9. The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein.

    Science.gov (United States)

    White, Michael R; Garcin, Elsa D

    2016-01-01

    The glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA (tRNA), the regulation of cellular messenger RNA (mRNA) stability and translation, as well as the regulation of replication and gene expression of many single-stranded RNA viruses. A growing body of evidence supports GAPDH-RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA-binding sequences. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function. WIREs RNA 2016, 7:53-70. doi: 10.1002/wrna.1315 For further resources related to this article, please visit the WIREs website.

  10. Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injur y

    Institute of Scientific and Technical Information of China (English)

    Guan Wang; Jian-ning Zhang; Jia-kui Guo; Ying Cai; Hong-sheng Sun; Kun Dong; Cheng-gang Wu

    2016-01-01

    Cold-inducible RNA-binding protein (CIRP), a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5°C on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP mRNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury.

  11. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  12. The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

    Science.gov (United States)

    Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

    2012-03-01

    The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

  13. TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues

    OpenAIRE

    Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

    2014-01-01

    microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents ...

  14. Cyclic AMP stimulates neurite outgrowth of lamprey reticulospinal neurons without substantially altering their biophysical properties.

    Science.gov (United States)

    Pale, T; Frisch, E B; McClellan, A D

    2013-08-15

    Reticulospinal (RS) neurons are critical for initiation of locomotor behavior, and following spinal cord injury (SCI) in the lamprey, the axons of these neurons regenerate and restore locomotor behavior within a few weeks. For lamprey RS neurons in culture, experimental induction of calcium influx, either in the growth cone or cell body, is inhibitory for neurite outgrowth. Following SCI, these neurons partially downregulate calcium channel expression, which would be expected to reduce calcium influx and possibly provide supportive conditions for axonal regeneration. In the present study, it was tested whether activation of second messenger signaling pathways stimulates neurite outgrowth of lamprey RS neurons without altering their electrical properties (e.g. spike broadening) so as to possibly increase calcium influx and compromise axonal growth. First, activation of cAMP pathways with forskolin or dbcAMP stimulated neurite outgrowth of RS neurons in culture in a PKA-dependent manner, while activation of cGMP signaling pathways with dbcGMP inhibited outgrowth. Second, neurophysiological recordings from uninjured RS neurons in isolated lamprey brain-spinal cord preparations indicated that dbcAMP or dbcGMP did not significantly affect any of the measured electrical properties. In contrast, for uninjured RS neurons, forskolin increased action potential duration, which might have increased calcium influx, but did not significantly affect most other electrical properties. Importantly, for injured RS neurons during the period of axonal regeneration, forskolin did not significantly alter their electrical properties. Taken together, these results suggest that activation of cAMP signaling by dbcAMP stimulates neurite outgrowth, but does not alter the electrical properties of lamprey RS neurons in such a way that would be expected to induce calcium influx. In conclusion, our results suggest that activation of cAMP pathways alone, without compensation for possible

  15. Effects of Chemotherapy-Induced Alterations in Cell Mechanical Properties on Cancer Metastasis

    Science.gov (United States)

    Prathivadhi, Sruti; Ekpenyong, Andrew; Nichols, Michael; Taylor, Carolyn; Ning, Jianhao

    Biological cells can modulate their mechanical properties to suit their functions and in response to changes in their environment. Thus, mechanical phenotyping of cells has been employed for tracking stem cell differentiation, bacterial infection, cell death, etc. Malignant transformation of cells also involves changes in mechanical properties. However, the extent to which mechanical properties of cancer cells contribute to metastasis is not well understood. Yet, more than 90% of all cancer deaths are directly related to metastasis. Transit of cells through the microcirculation is one of the key features of metastasis. We hypothesize that cancer treatment regimens do inadvertently alter cell mechanical properties in ways that might promote cancer metastasis. We use a microfluidic microcirculation mimetic (MMM) platform which mimics the capillary constrictions of the pulmonary and peripheral microcirculation to determine if in-vivo-like mechanical stimuli can evoke different responses from cells subjected to various cancer drugs. In particular, we show that cancer cells treated with chemotherapeutic drugs such as daunorubicin, become more deformable at short timescales (0.1 s) and transit faster through the device. Our results are first steps in evaluating the pro- or anti-metastatic effects of chemotherapeutic drugs based on their induced alterations in cell mechanical properties.

  16. Genetic variation and RNA binding proteins: tools and techniques to detect functional polymorphisms.

    Science.gov (United States)

    Soemedi, Rachel; Vega, Hugo; Belmont, Judson M; Ramachandran, Sohini; Fairbrother, William G

    2014-01-01

    At its most fundamental level the goal of genetics is to connect genotype to phenotype. This question is asked at a basic level evaluating the role of genes and pathways in genetic model organism. Increasingly, this question is being asked in the clinic. Genomes of individuals and populations are being sequenced and compared. The challenge often comes at the stage of analysis. The variant positions are analyzed with the hope of understanding human disease. However after a genome or exome has been sequenced, the researcher is often deluged with hundreds of potentially relevant variations. Traditionally, amino-acid changing mutations were considered the tractable class of disease-causing mutations; however, mutations that disrupt noncoding elements are the subject of growing interest. These noncoding changes are a major avenue of disease (e.g., one in three hereditary disease alleles are predicted to affect splicing). Here, we review some current practices of medical genetics, the basic theory behind biochemical binding and functional assays, and then explore technical advances in how variations that alter RNA protein recognition events are detected and studied. These advances are advances in scale-high-throughput implementations of traditional biochemical assays that are feasible to perform in any molecular biology laboratory. This chapter utilizes a case study approach to illustrate some methods for analyzing polymorphisms. The first characterizes a functional intronic SNP that deletes a high affinity PTB site using traditional low-throughput biochemical and functional assays. From here we demonstrate the utility of high-throughput splicing and spliceosome assembly assays for screening large sets of SNPs and disease alleles for allelic differences in gene expression. Finally we perform three pilot drug screens with small molecules (G418, tetracycline, and valproic acid) that illustrate how compounds that rescue specific instances of differential pre-mRNA processing

  17. The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema; Hall, Traci M. Tanaka; Gavis, Elizabeth R.

    2017-04-01

    The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subset of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.

  18. Interactions among rsmX ncRNAs and Rsm RNA-binding proteins in the plant pathogen Pseudomonas syringae DC3000

    Science.gov (United States)

    In response to changing environmental stimuli, many bacterial species utilize the Csr/Rsm system of posttranscriptional gene expression regulation to control metabolism, motility, biofilm formation, and quorum sensing. Most Csr/Rsm RNA binding proteins are thought to bind near the 5’ end of mRNA tra...

  19. Alteration kinetics of a simplified nuclear glass in an aqueous medium: effects of solution chemistry and of protective gel properties on diminishing the alteration rate

    Science.gov (United States)

    Jégou, C.; Gin, S.; Larché, F.

    2000-07-01

    The alteration kinetics of the French SON 68 nuclear glass simplified to its three major constituent elements (Si, B and Na) were investigated by static experiments at 90°C in order to deconvolute the effects of the solution chemistry and of the protective properties of the alteration gel on the diminishing alteration rate over time. A glass dissolution experiment in static conditions showed that the initial rate r0 was maintained even after silicon saturation of the solution. As the reaction progressed, the glass alteration rate gradually diminished over time. These results show that the driving force behind the alteration of this glass cannot be defined by the difference from saturation with respect to amorphous silica, and that reaching saturation is not a criterion for the end of alteration. The drop in the dissolution rate observed at a high degree of reaction progress is correlated with the formation of the silica gel that develops at the glass/solution interface. Confronting the experimental data with a model taking into account a diffusion boundary layer shows that the conventional tools of chemical thermodynamics are ill adapted to describing the formation and development of the silica gel layer over time. This study reveals that only a dynamic process of hydrolysis and condensation of silicon at the glass/gel interface can account for the formation of the gel layer. The glass alteration rate under silica saturation conditions would thus be highly dependent on the silicon recondensation rate in this `dynamic percolation' concept.

  20. Involvement of XZFP36L1,an RNA-binding protein,in Xenopus neural development%Involvement of XZFP36L1, an RNA-binding protein,in Xenopus neural development

    Institute of Scientific and Technical Information of China (English)

    Yingjie XIA; Shuhua ZHAO; Bingyu MAO

    2012-01-01

    Xenopus ZFP36L1 (zinc finger protein 36,C3H type-like 1) belongs to the ZFP36 family of RNA-binding proteins,which contains two characteristic tandem CCCH-type zinc-finger domains.The ZFP36 proteins can bind AU-rich elements in 3' untranslated regions of target mRNAs and promote their turnover.However,the expression and role of ZFP36 genes during neural development in Xenopus embryos remains largely tmknown.The present study showed that Xenopus ZFP36L1 was expressed at the dorsal part of the forebrain,forebrain-midbrain boundary,and midbrain-hindbrain boundary from late neurula stages to tadpole stages of embryonic development.Overexpression of XZFP36L1 in Xenopus embryos inhibited neural induction and differentiation,leading to severe neural tube defects.The function of XZP36L1 requires both its zinc finger and C terminal domains,which also affect its subcellular localization.These results suggest that XZFP36L1 is likely involved in neural development in Xenopus and might play an important role in post-transcriptional regulation.

  1. APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

    Directory of Open Access Journals (Sweden)

    Katia Marrocco

    Full Text Available BACKGROUND: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. CONCLUSIONS/SIGNIFICANCE: Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular

  2. Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics

    Science.gov (United States)

    Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

    2017-02-01

    Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process.

  3. Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb; Lu, Gang; Bigler, Rebecca; Jezyk, Mark R.; Li, Chunhua; Tanaka Hall, Traci M.; Wang, Zefeng (NIH); (Beijing U); (UNC)

    2011-10-28

    Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

  4. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis

    Science.gov (United States)

    Yang, Leiyun; Yang, Fen; Wang, Yi; Zhu, Jian-Kang; Hua, Jian

    2016-01-01

    Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance. PMID:27138552

  5. The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora

    Science.gov (United States)

    Ancona, Veronica; Lee, Jae Hoon; Zhao, Youfu

    2016-01-01

    The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA. PMID:27845410

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

    Science.gov (United States)

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

    2015-08-11

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

  7. The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora.

    Science.gov (United States)

    Ancona, Veronica; Lee, Jae Hoon; Zhao, Youfu

    2016-11-15

    The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA.

  8. The RNA-binding protein Spo5 promotes meiosis II by regulating cyclin Cdc13 in fission yeast.

    Science.gov (United States)

    Arata, Mayumi; Sato, Masamitsu; Yamashita, Akira; Yamamoto, Masayuki

    2014-03-01

    Meiosis comprises two consecutive nuclear divisions, meiosis I and II. Despite this unique progression through the cell cycle, little is known about the mechanisms controlling the sequential divisions. In this study, we carried out a genetic screen to identify factors that regulate the initiation of meiosis II in the fission yeast Schizosaccharomyces pombe. We identified mutants deficient in meiosis II progression and repeatedly isolated mutants defective in spo5, which encodes an RNA-binding protein. Using fluorescence microscopy to visualize YFP-tagged protein, we found that spo5 mutant cells precociously lost Cdc13, the major B-type cyclin in fission yeast, before meiosis II. Importantly, the defect in meiosis II was rescued by increasing CDK activity. In wild-type cells, cdc13 transcripts increased during meiosis II, but this increase in cdc13 expression was weaker in spo5 mutants. Thus, Spo5 is a novel regulator of meiosis II that controls the level of cdc13 expression and promotes de novo synthesis of Cdc13. We previously reported that inhibition of Cdc13 degradation is necessary to initiate meiosis II; together with the previous information, the current findings indicate that the dual control of Cdc13 by de novo synthesis and suppression of proteolysis ensures the progression of meiosis II.

  9. Analysis of the Microprocessor in Dictyostelium: The Role of RbdB, a dsRNA Binding Protein.

    Science.gov (United States)

    Meier, Doreen; Kruse, Janis; Buttlar, Jann; Friedrich, Michael; Zenk, Fides; Boesler, Benjamin; Förstner, Konrad U; Hammann, Christian; Nellen, Wolfgang

    2016-06-01

    We identified the dsRNA binding protein RbdB as an essential component in miRNA processing in Dictyostelium discoideum. RbdB is a nuclear protein that accumulates, together with Dicer B, in nucleolar foci reminiscent of plant dicing bodies. Disruption of rbdB results in loss of miRNAs and accumulation of primary miRNAs. The phenotype can be rescued by ectopic expression of RbdB thus allowing for a detailed analysis of domain function. The lack of cytoplasmic dsRBD proteins involved in miRNA processing, suggests that both processing steps take place in the nucleus thus resembling the plant pathway. However, we also find features e.g. in the domain structure of Dicer which suggest similarities to animals. Reduction of miRNAs in the rbdB- strain and their increase in the Argonaute A knock out allowed the definition of new miRNAs one of which appears to belong to a new non-canonical class.

  10. Double-Stranded RNA-Binding Protein 4 Is Required for Resistance Signaling against Viral and Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Shifeng Zhu

    2013-09-01

    Full Text Available Plant viruses often encode suppressors of host RNA silencing machinery, which occasionally function as avirulence factors that are recognized by host resistance (R proteins. For example, the Arabidopsis R protein, hypersensitive response to TCV (HRT, recognizes the turnip crinkle virus (TCV coat protein (CP. HRT-mediated resistance requires the RNA-silencing component double-stranded RNA-binding protein 4 (DRB4 even though it neither is associated with the accumulation of TCV-specific small RNA nor requires the RNA silencing suppressor function of CP. HRT interacts with the cytosolic fraction of DRB4. Interestingly, TCV infection both increases the cytosolic DRB4 pool and inhibits the HRT-DRB4 interaction. The virulent R8A CP derivative, which induces a subset of HRT-derived responses, also disrupts this interaction. The differential localization of DRB4 in the presence of wild-type and R8A CP implies the importance of subcellular compartmentalization of DRB4. The requirement of DRB4 in resistance to bacterial infection suggests a universal role in R-mediated defense signaling.

  11. Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

    Science.gov (United States)

    Vian, A.; Henry-Vian, C.; Davies, E.

    1999-01-01

    It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

  12. Drosophila Shep and C. elegans SUP-26 are RNA-binding proteins that play diverse roles in nervous system development.

    Science.gov (United States)

    Schachtner, Logan T; Sola, Ismail E; Forand, Daniel; Antonacci, Simona; Postovit, Adam J; Mortimer, Nathan T; Killian, Darrell J; Olesnicky, Eugenia C

    2015-11-01

    The Caenorhabditis elegans gene sup-26 encodes a well-conserved RNA-recognition motif-containing RNA-binding protein (RBP) that functions in dendrite morphogenesis of the PVD sensory neuron. The Drosophila ortholog of sup-26, alan shepard (shep), is expressed throughout the nervous system and has been shown to regulate neuronal remodeling during metamorphosis. Here, we extend these studies to show that sup-26 and shep are required for the development of diverse cell types within the nematode and fly nervous systems during embryonic and larval stages. We ascribe roles for sup-26 in regulating dendrite number and the expression of genes involved in mechanosensation within the nematode peripheral nervous system. We also find that in Drosophila, shep regulates dendrite length and branch order of nociceptive neurons, regulates the organization of neuronal clusters of the peripheral nervous system and the organization of axons within the ventral nerve cord. Taken together, our results suggest that shep/sup-26 orthologs play diverse roles in neural development across animal species. Moreover, we discuss potential roles for shep/sup-26 orthologs in the human nervous system.

  13. Multiple Functions of the RNA-Binding Protein HuR in Cancer Progression, Treatment Responses and Prognosis

    Directory of Open Access Journals (Sweden)

    Baocheng Wang

    2013-05-01

    Full Text Available The human embryonic lethal abnormal vision-like protein, HuR, is a member of the Hu family of RNA-binding proteins. Over the past decade, this ubiquitously expressed protein has been extensively investigated in cancer research because it is involved in the regulation of mRNA stability and translation in many cell types. HuR activity and function is associated with its subcellular distribution, transcriptional regulation, translational and post-translational modifications. HuR regulation of target mRNAs is based on the interaction between the three specific domains of HuR protein and one or several U- or AU-rich elements (AREs in the untranslated region of target mRNAs. A number of cancer-related transcripts containing AREs, including mRNAs for proto-oncogenes, cytokines, growth factors, and invasion factors, have been characterized as HuR targets. It has been proposed that HuR has a central tumorigenic activity by enabling multiple cancer phenotypes. In this review, we comprehensively survey the existing evidence with regard to the diverse functions of HuR in caner development and progression. The current data also suggest that HuR might be a novel and promising therapeutic target and a marker for treatment response and prognostic evaluation.

  14. Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities.

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    Liang, L; Diehl-Jones, W; Lasko, P

    1994-05-01

    The Drosophila gene vasa encodes a DEAD-box protein, which is localized during early oogenesis to the perinuclear region of the nurse cells and later to the pole plasm at the posterior end of the oocyte. Posterior localization of vasa protein depends upon the functions of four genes: capu, spir, osk and stau. We have found that localization of vasa to the perinuclear nuage is abolished in most vas alleles, but is unaffected by mutations in four genes required upstream for its pole plasm localization. Thus localization of vasa to the nuage particles is independent of the pole plasm assembly pathway. Furthermore, electron-dense nuage particles are less abundant in the cytoplasm of nurse cells from vas mutants that fail to exhibit perinuclear localization, suggesting that the formation of the nuage depends upon vas function. Eight of nine vas point mutations cause codon substitutions in a region conserved among DEAD-box genes. The proteins from two mutant alleles that retain the capacity to localize to the posterior pole of the oocyte, vasO14 and vasO11, are both severely reduced in RNA-binding and -unwinding activity as compared to the wild-type protein on a variety of RNA substrates including in vitro synthesized pole plasm RNAs. Initial recruitment of vasa to the pole plasm must consequently depend upon protein-protein interactions but, once localized, vasa must bind to RNA to mediate germ cell formation.

  15. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Keryer-Bibens, Cecile, E-mail: cecile.keryer-bibens@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Legagneux, Vincent; Namanda-Vanderbeken, Allen [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Cosson, Bertrand [UPMC Universite de Paris 06, UMR 7150, Equipe Traduction Cycle Cellulaire et Developpement, Station Biologique de Roscoff, 29682 Roscoff (France); CNRS, UMR 7150, Station Biologique de Roscoff, 29682 Roscoff (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Paillard, Luc [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Poncet, Didier [Virologie Moleculaire et Structurale, UMR CNRS, 2472, INRA, 1157, 91198 Gif sur Yvette (France); Osborne, H. Beverley, E-mail: beverley.osborne@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France)

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  16. Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.

    Science.gov (United States)

    Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb; Lu, Gang; Bigler, Rebecca; Jezyk, Mark R; Li, Chunhua; Hall, Traci M Tanaka; Wang, Zefeng

    2011-07-29

    Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

  17. Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

    Science.gov (United States)

    Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi

    2003-05-15

    We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.

  18. Saturated fatty acids alter the late secretory pathway by modulating membrane properties.

    Science.gov (United States)

    Payet, Laurie-Anne; Pineau, Ludovic; Snyder, Ellen C R; Colas, Jenny; Moussa, Ahmed; Vannier, Brigitte; Bigay, Joelle; Clarhaut, Jonathan; Becq, Frédéric; Berjeaud, Jean-Marc; Vandebrouck, Clarisse; Ferreira, Thierry

    2013-12-01

    Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic β-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

  19. Noninvasive induction implant heating: an approach for contactless altering of mechanical properties of shape memory implants.

    Science.gov (United States)

    Pfeifer, Ronny; Hustedt, Michael; Wesling, Volker; Hurschler, Christoph; Olender, Gavin; Mach, Martin; Gösling, Thomas; Müller, Christian W

    2013-01-01

    This article shows an approach to change the properties of an orthopaedic shape memory implant within biological tissue, using contactless induction heating. Due to inducing the one way-memory effect, triggered by the rise of temperature within the implant, the geometry and hence the mechanical properties of the implant itself, are altered. The power uptake of the implant, depending on the induction parameters as well as on its position within the induction coil, is shown. Thermographic measurements are carried out in order to determine the surface temperature distribution of the implant. In order to simulate biological tissue, the implant was embedded in agarose gel. Suitable heating parameters, in terms of a short heating process in combination with a reduced heat impact on the surrounding environment, were determined.

  20. Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

    Directory of Open Access Journals (Sweden)

    Dever Thomas E

    2008-03-01

    Full Text Available Abstract Background Double-stranded (ds RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs. Fish and amphibian PKR genes have not been described so far. Results Here we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2α in yeast. Conclusion Considering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both ds

  1. Altered goblet cell differentiation and surface mucus properties in Hirschsprung disease.

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    Jay R Thiagarajah

    Full Text Available Hirschsprung disease-associated enterocolitis (HAEC leads to significant mortality and morbidity, but its pathogenesis remains unknown. Changes in the colonic epithelium related to goblet cells and the luminal mucus layer have been postulated to play a key role. Here we show that the colonic epithelium of both aganglionic and ganglionic segments are altered in patients and in mice with Hirschsprung disease (HSCR. Structurally, goblet cells were altered with increased goblet cell number and reduced intracellular mucins in the distal colon of biopsies from patients with HSCR. Endothelin receptor B (Ednrb mutant mice showed increased goblet cell number and size and increased cell proliferation compared to wild-type mice in aganglionic segments, and reduced goblet cell size and number in ganglionic segments. Functionally, compared to littermates, Ednrb-/- mice showed increased transepithelial resistance, reduced stool water content and similar chloride secretion in the distal colon. Transcript levels of goblet cell differentiation factors SPDEF and Math1 were increased in the distal colon of Ednrb-/- mice. Both distal colon from Ednrb mice and biopsies from HSCR patients showed reduced Muc4 expression as compared to controls, but similar expression of Muc2. Particle tracking studies showed that mucus from Ednrb-/- mice provided a more significant barrier to diffusion of 200 nm nanoparticles as compared to wild-type mice. These results suggest that aganglionosis is associated with increased goblet cell proliferation and differentiation and subsequent altered surface mucus properties, prior to the development of inflammation in the distal colon epithelium. Restoration of normal goblet cell function and mucus layer properties in the colonic epithelium may represent a therapeutic strategy for prevention of HAEC.

  2. Preacclimation alters Salmonella Enteritidis surface properties and its initial attachment to food contact surfaces.

    Science.gov (United States)

    Yang, Yishan; Kumar, Amit; Zheng, Qianwang; Yuk, Hyun-Gyun

    2015-04-01

    Exposure of Salmonella to environmental stress, prior to its adherence to a food contact surface, may change the cell surface properties and consequently affect its initial attachment and biofilm formation. This study investigated the influence of temperature and pH preacclimation on the initial attachment of Salmonella Enteritidis to acrylic and stainless steel. Besides, changes in physicochemical properties of cells were examined; and their surface attachment was modeled by xDLVO theory. Results showed that control cells pre-grown at 37°C had significantly (P0.05) different from control cells pre-grown at pH 7.3, but they were significantly higher compared to cells pre-grown at pH 8.3 and 9.0. No significant difference was observed between cell attachment to acrylic and stainless steel, although they had different physicochemical properties. The xDLVO theory successfully explained higher attachment for cells pre-grown at optimal condition on both contact surfaces. However, the xDLVO theory could not explain the similar attachment of cells to acrylic and stainless steel. This study elucidates that commonly used intervention technologies including cold storage, thermal treatment, and alkaline antimicrobial agents might alter the physicochemical properties of S. Enteritidis cells and result in varied initial attachment levels.

  3. Cold-inducible RNA-binding protein is an important mediator of alcohol-induced brain inflammation.

    Directory of Open Access Journals (Sweden)

    Salil R Rajayer

    Full Text Available Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP, a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP(-/- mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP(-/- mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP(-/- mice. In cultured BV2 cells (mouse microglia, ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.

  4. Brassica RNA binding protein ERD4 is involved in conferring salt, drought tolerance and enhancing plant growth in Arabidopsis.

    Science.gov (United States)

    Rai, Archana N; Tamirisa, Srinath; Rao, K V; Kumar, Vinay; Suprasanna, P

    2016-03-01

    'Early responsive to dehydration' (ERD) genes are a group of plant genes having functional roles in plant stress tolerance and development. In this study, we have isolated and characterized a Brassica juncea 'ERD' gene (BjERD4) which encodes a novel RNA binding protein. The expression pattern of ERD4 analyzed under different stress conditions showed that transcript levels were increased with dehydration, sodium chloride, low temperature, heat, abscisic acid and salicylic acid treatments. The BjERD4 was found to be localized in the chloroplasts as revealed by Confocal microscopy studies. To study the function, transgenic Arabidopsis plants were generated and analyzed for various morphological and physiological parameters. The overexpressing transgenic lines showed significant increase in number of leaves with more leaf area and larger siliques as compared to wild type plants, whereas RNAi:ERD4 transgenic lines showed reduced leaf number, leaf area, dwarf phenotype and delayed seed germination. Transgenic Arabidopsis plants overexpressing BjERD4 gene also exhibited enhanced tolerance to dehydration and salt stresses, while the knockdown lines were susceptible as compared to wild type plants under similar stress conditions. It was observed that BjERD4 protein could bind RNA as evidenced by the gel-shift assay. The overall results of transcript analysis, RNA gel-shift assay, and transgenic expression, for the first time, show that the BjERD4 is involved in abiotic stress tolerance besides offering new clues about the possible roles of BjERD4 in plant growth and development.

  5. The RNA-binding protein PCBP2 facilitates gastric carcinoma growth by targeting miR-34a

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Cheng-En; Liu, Yong-Chao [Department of General Surgery, Huashan Hospital, Fudan University, Shanghai (China); Zhang, Hui-Dong [Department of General Surgery, Shanghai Children’s Medical Center, Shanghai (China); Huang, Guang-Jian, E-mail: huanggjfdu@sina.com [Department of General Surgery, Huashan Hospital, Fudan University, Shanghai (China)

    2014-06-13

    Highlights: • PCBP2 is overexpressed in human gastric cancer. • PCBP2 high expression predicts poor survival. • PCBP2 regulates gastric cancer growth in vitro and in vivo. • PCBP2 regulates gastric cancer apoptosis by targeting miR-34a. - Abstract: Gastric carcinoma is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. However, the mechanism underling gastric cancer is still not fully understood. Here in the present study, we identify the RNA-binding protein PCBP2 as an oncogenic protein in human gastric carcinoma. Our results show that PCBP2 is up-regulated in human gastric cancer tissues compared to adjacent normal tissues, and that high level of PCBP2 predicts poor overall and disease-free survival. Knockdown of PCBP2 in gastric cancer cells inhibits cell proliferation and colony formation in vitro, whereas opposing results are obtained when PCBP2 is overexpressed. Our in vivo subcutaneous xenograft results also show that PCBP2 can critically regulate gastric cancer cell growth. In addition, we find that PCBP2-depletion induces apoptosis in gastric cancer cells via up-regulating expression of pro-apoptotic proteins and down-regulating anti-apoptotic proteins. Mechanically, we identify that miR-34a as a target of PCBP2, and that miR-34a is critically essential for the function of PCBP2. In summary, PCBP2 promotes gastric carcinoma development by regulating the level of miR-34a.

  6. Long-term memory consolidation: The role of RNA-binding proteins with prion-like domains.

    Science.gov (United States)

    Sudhakaran, Indulekha P; Ramaswami, Mani

    2016-10-11

    Long-term and short-term memories differ primarily in the duration of their retention. At a molecular level, long-term memory (LTM) is distinguished from short-term memory (STM) by its requirement for new gene expression. In addition to transcription (nuclear gene expression) the translation of stored mRNAs is necessary for LTM formation. The mechanisms and functions for temporal and spatial regulation of mRNAs required for LTM is a major contemporary problem, of interest from molecular, cell biological, neurobiological and clinical perspectives. This review discusses primary evidence in support for translational regulatory events involved in LTM and a model in which different phases of translation underlie distinct phases of consolidation of memories. However, it focuses largely on mechanisms of memory persistence and the role of prion-like domains in this defining aspect of long-term memory. We consider primary evidence for the concept that Cytoplasmic Polyadenylation Element Binding (CPEB) protein enables the persistence of formed memories by transforming in prion-like manner from a soluble monomeric state to a self-perpetuating and persistent polymeric translationally active state required for maintaining persistent synaptic plasticity. We further discuss prion-like domains prevalent on several other RNA-binding proteins involved in neuronal translational control underlying LTM. Growing evidence indicates that such RNA regulatory proteins are components of mRNP (RiboNucleoProtein) granules. In these proteins, prion-like domains, being intrinsically disordered, could mediate weak transient interactions that allow the assembly of RNP granules, a source of silenced mRNAs whose translation is necessary for LTM. We consider the structural bases for RNA granules formation as well as functions of disordered domains and discuss how these complicate the interpretation of existing experimental data relevant to general mechanisms by which prion-domain containing RBPs

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

    Science.gov (United States)

    Eberhardt, Wolfgang; Doller, Anke; Pfeilschifter, Josef

    2012-06-01

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

  8. [Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center].

    Science.gov (United States)

    Bausk, E V; Graĭfer, D M; Karpova, G G

    1985-01-01

    Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.

  9. The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system.

    Science.gov (United States)

    Rogulja-Ortmann, Ana; Picao-Osorio, Joao; Villava, Casandra; Patraquim, Pedro; Lafuente, Elvira; Aspden, Julie; Thomsen, Stefan; Technau, Gerhard M; Alonso, Claudio R

    2014-05-01

    The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

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

    Science.gov (United States)

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

    2016-07-01

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

  11. A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol.

    Science.gov (United States)

    Anji, Antje; Kumari, Meena

    2006-05-01

    Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half-life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis-acting region(s) within the NR1 3'-untranslated region (UTR) and identify NR1 3'-UTR binding trans-acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156-nt cis-acting region that binds to polysomal trans-acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis-trans interactions. Importantly, the 3'-UTR binding activity was significantly up-regulated in the presence of ethanol. UV cross-link analysis detected three NR1 3'-UTR binding proteins and their molecular mass calculated by Northwestern analysis was approximately 88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up-regulation of the 88-kDa protein after chronic ethanol treatment. The 88-kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIbeta). That GIIbeta is indeed a trans-acting protein and binds specifically to 3'-UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT-PCR. Western blotting data established a significant increase of GIIbeta polypeptide in chronic ethanol-exposed fetal cortical neurons. We hypothesize that the identified cis-acting region and the associated RNA-binding proteins are important regulators of NR1 subunit gene expression.

  12. Expression of Progesterone Receptor Membrane Component 1 (PGRMC1, Progestin and AdipoQ Receptor 7 (PAQPR7, and Plasminogen Activator Inhibitor 1 RNA-Binding Protein (PAIRBP1 in Glioma Spheroids In Vitro

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    Juraj Hlavaty

    2016-01-01

    Full Text Available Objective. Some effects of progesterone on glioma cells can be explained through the slow, genomic mediated response via nuclear receptors; the other effects suggest potential role of a fast, nongenomic action mediated by membrane-associated progesterone receptors. Methods. The effects of progesterone treatment on the expression levels of progesterone receptor membrane component 1 (PGRMC1, plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1, and progestin and adipoQ receptor 7 (PAQR7 on both mRNA and protein levels were investigated in spheroids derived from human glioma cell lines U-87 MG and LN-229. Results. The only significant alteration at the transcript level was the decrease in PGRMC1 mRNA observed in LN-229 spheroids treated with 30 ng/mL of progesterone. No visible alterations at the protein levels were observed using immunohistochemical analysis. Stimulation of U-87 MG spheroids resulted in an increase of PGRMC1 but a decrease of PAIRBP1 protein. Double immunofluorescent detection of PGRMC1 and PAIRBP1 identified the two proteins to be partially colocalized in the cells. Western blot analysis revealed the expected bands for PGRMC1 and PAIRBP1, whereas two bands were detected for PAQR7. Conclusion. The progesterone action is supposed to be mediated via membrane-associated progesterone receptors as the nuclear progesterone receptor was absent in tested spheroids.

  13. RNA binding proteins hnRNP A2/B1 and CUGBP1 suppress Fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS

    OpenAIRE

    Sofola, Oyinkan A.; Jin, Peng; QIN, YUNLONG; Duan, Ranhui; LIU, Huijie; de Haro, Maria; Nelson,David L.; Botas, Juan

    2007-01-01

    Fragile X associated tremor ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile-X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA mediated neurodegenerative disease caused by the titration of RNA binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses premutation length repeat (90 CGG repeats)...

  14. Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells

    OpenAIRE

    Zhe Geng; Ping Li; Li Tan; Houyan Song

    2015-01-01

    RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression...

  15. The RNA-binding protein TIAR is translocated from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death.

    OpenAIRE

    Taupin, J L; Tian, Q.; Kedersha, N; Robertson, M.; Anderson, P

    1995-01-01

    We have determined the structure, intracellular localization, and tissue distribution of TIAR, a TIA-1-related RNA-binding protein. Two related isoforms of TIAR, migrating at 42 and 50 kDa, are expressed in primate cells. Unlike TIA-1, which is found in the granules of cytotoxic lymphocytes, TIAR is concentrated in the nucleus of hematopoietic and nonhematopoietic cells. Because TIAR can trigger DNA fragmentation in permeabilized thymocytes, it is a candidate effector of apoptotic cell death....

  16. Conjugation of D-glucosamine to bovine trypsin increases thermal stability and alters functional properties.

    Science.gov (United States)

    Gizurarson, Jóhann Grétar Kröyer; Filippusson, Hörður

    2015-01-01

    D-Glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 D-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T50% was increased by 7 °C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25 °C (without stabilizing Ca(2+) ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (k(cat)/K(m)) than unmodified trypsin against the substrate N-α-benzoyl-L-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate.

  17. The RNA binding protein ESRP1 fine-tunes the expression of pluripotency-related factors in mouse embryonic stem cells.

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    Sharmila Fagoonee

    Full Text Available In pluripotent stem cells, there is increasing evidence for crosstalk between post-transcriptional and transcriptional networks, offering multifold steps at which pluripotency can be controlled. In addition to well-studied transcription factors, chromatin modifiers and miRNAs, RNA-binding proteins are emerging as fundamental players in pluripotency regulation. Here, we report a new role for the RNA-binding protein ESRP1 in the control of pluripotency. Knockdown of Esrp1 in mouse embryonic stem cells induces, other than the well-documented epithelial to mesenchymal-like state, also an increase in expression of the core transcription factors Oct4, Nanog and Sox2, thereby enhancing self-renewal of these cells. Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells. We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading. ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. Importantly, both mouse and human epithelial stem cells highly express ESRP1, pinpointing the importance of this RNA-binding protein in stem cell biology.

  18. RNA Binding Proteins RZ-1B and RZ-1C Play Critical Roles in Regulating Pre-mRNA Splicing and Gene Expression during Development in Arabidopsis.

    Science.gov (United States)

    Wu, Zhe; Zhu, Danling; Lin, Xiaoya; Miao, Jin; Gu, Lianfeng; Deng, Xian; Yang, Qian; Sun, Kangtai; Zhu, Danmeng; Cao, Xiaofeng; Tsuge, Tomohiko; Dean, Caroline; Aoyama, Takashi; Gu, Hongya; Qu, Li-Jia

    2016-01-01

    Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here, we report the functions of two proteins containing RNA recognition motifs, RZ-1B and RZ-1C, in Arabidopsis thaliana. RZ-1B and RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C termini. RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA binding activity of RZ-1C with SR proteins through overexpression of the C terminus of RZ-1C conferred defective phenotypes similar to those observed in rz-1b rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of RZ-1B and RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that RZ-1C directly targeted FLOWERING LOCUS C (FLC), promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins.

  19. The RNA-binding proteins FMR1, rasputin and caprin act together with the UBA protein lingerer to restrict tissue growth in Drosophila melanogaster.

    Science.gov (United States)

    Baumgartner, Roland; Stocker, Hugo; Hafen, Ernst

    2013-01-01

    Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer. Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex, respectively, has been well documented. Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear. Here, we identify Lingerer (Lig), an UBA domain-containing protein, as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 (FMR1) and Caprin (Capr) and directly interacts with and regulates the RNA-binding protein Rasputin (Rin) in Drosophila melanogaster. lig mutant organs overgrow due to increased proliferation, and a reporter for the JAK/STAT signaling pathway is upregulated in a lig mutant situation. rin, Capr or FMR1 in combination as double mutants, but not the respective single mutants, display lig like phenotypes, implicating a redundant function of Rin, Capr and FMR1 in growth control in epithelial tissues. Thus, Lig regulates cell proliferation during development in concert with Rin, Capr and FMR1.

  20. The RNA-binding proteins FMR1, rasputin and caprin act together with the UBA protein lingerer to restrict tissue growth in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Roland Baumgartner

    Full Text Available Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer. Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex, respectively, has been well documented. Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear. Here, we identify Lingerer (Lig, an UBA domain-containing protein, as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 (FMR1 and Caprin (Capr and directly interacts with and regulates the RNA-binding protein Rasputin (Rin in Drosophila melanogaster. lig mutant organs overgrow due to increased proliferation, and a reporter for the JAK/STAT signaling pathway is upregulated in a lig mutant situation. rin, Capr or FMR1 in combination as double mutants, but not the respective single mutants, display lig like phenotypes, implicating a redundant function of Rin, Capr and FMR1 in growth control in epithelial tissues. Thus, Lig regulates cell proliferation during development in concert with Rin, Capr and FMR1.

  1. Vector-averaged gravity does not alter acetylcholine receptor single channel properties

    Science.gov (United States)

    Reitstetter, R.; Gruener, R.

    1994-01-01

    To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

  2. Alteration of time-resolved autofluorescence properties of rat aorta, induced by diabetes mellitus

    Science.gov (United States)

    Uherek, M.; Uličná, O.; Vančová, O.; Muchová, J.; Ďuračková, Z.; Šikurová, L.; Chorvát, D.

    2016-10-01

    Changes in autofluorescence properties of isolated rat aorta, induced by diabetes mellitus, were detected using time-resolved fluorescence spectroscopy with pulsed ultraviolet (UV) laser excitation. We demonstrated that time-resolved spectroscopy was able to detect changes in aorta tissues related to diabetes and unambiguously discriminate diabetic (τ 1 0.63  ±  0.05 ns, τ 2 3.66  ±  0.10 ns) samples from the control (τ 1 0.76  ±  0.03 ns, τ 2 4.48  ±  0.15 ns) group. We also report changes in the ratio of relative amplitudes of the two lifetime component in aorta tissue during diabetes, most likely related to the pseudohypoxic state with altered NADH homeostasis.

  3. Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers.

    Science.gov (United States)

    Burga, Laura N; Tung, Nadine M; Troyan, Susan L; Bostina, Mihnea; Konstantinopoulos, Panagiotis A; Fountzilas, Helena; Spentzos, Dimitrios; Miron, Alexander; Yassin, Yosuf A; Lee, Bernard T; Wulf, Gerburg M

    2009-02-15

    Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MEC) with altered proliferation and differentiation properties. Using a three-dimensional culture technique to grow MECs ex vivo, we found that the ability to form colonies, an indication of clonality, was restricted to the aldehyde dehydrogenase 1-positive fraction in MECs but not in HCC1937 BRCA1-mutant cancer cells. Primary MECs from BRCA1 mutation carriers (n = 9) had a 28% greater ability for clonal growth compared with normal controls (n = 6; P = 0.006), and their colonies were significantly larger. Colonies in controls and BRCA1 mutation carriers stained positive for BRCA1 by immunohistochemistry, and 79% of the examined single colonies from BRCA1 carriers retained heterozygosity for BRCA1 (ROH). Colonies from BRCA1 mutation carriers frequently showed high epidermal growth factor receptor (EGFR) expression (71% EGFR positive versus 44% in controls) and were negative for estrogen receptor (ERalpha; 32% ER negative, 44% mixed, 24% ER positive versus 90% ER positive in controls). Expression of CK14 and p63 were not significantly different. Microarray studies revealed that colonies from BRCA1-mutant PMECs anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is up-regulated. We conclude that BRCA1 haploinsufficiency leads to an increased ability for clonal growth and proliferation in the PMECs of BRCA1 mutation carriers, possibly as a result of EGFR pathway activation. These altered growth and differentiation properties may render BRCA1-mutant PMECs vulnerable to transformation and predispose to the development of ER-negative, EGFR-positive breast cancers.

  4. Hydrothermal alteration of surficial rocks at Solfatara (Campi Flegrei): Petrophysical properties and implications for phreatic eruption processes

    Science.gov (United States)

    Mayer, Klaus; Scheu, Bettina; Montanaro, Cristian; Yilmaz, Tim I.; Isaia, Roberto; Aßbichler, Donjá; Dingwell, Donald B.

    2016-06-01

    Solfatara crater is located within the Campi Flegrei caldera to the west of Naples (Italy). It is one of the largest fumarolic manifestations known, and the rocks hosting the hydrothermal system are affected by intense hydrothermal alteration. Alteration can result in changes of degassing behavior, and in the formation of a cap rock thereby increasing the probability of phreatic eruptions. Here, we investigate the effects of alunitic (solfataric) alteration on the mineralogy, the physical properties (porosity, density, permeability) and the mechanical properties (strength) of the rocks involved, as well as its influence on fragmentation and ejection behavior. Our results show that the pristine mineralogy of deposits from the vicinity of the Solfatara cryptodome and from Pisciarelli is almost completely replaced by amorphous silica and alunite. The differences in the degree of alteration among the samples series are reflected in the investigated properties and behavior as well as in the analysis of the experimentally generated particles. Alunitic alteration increases porosity and permeability, whereas it reduces density, elastic wave velocity and strength leading to higher fragmentation and ejection speeds for the sample series examined in this study. Our results also show that alteration results in the generation of a high fraction of fines (particle sizes < 10 μm) during fragmentation, mainly composed of alunite crystals. Due to their potential for inducing chronic disease, dispersion of such material should represent a serious health hazard on a local scale and the evaluation of precautions should be considered.

  5. A deficiency in cold-inducible RNA-binding protein accelerates the inflammation phase and improves wound healing.

    Science.gov (United States)

    Idrovo, Juan Pablo; Jacob, Asha; Yang, Weng Lang; Wang, Zhimin; Yen, Hao Ting; Nicastro, Jeffrey; Coppa, Gene F; Wang, Ping

    2016-02-01

    Chronic or non-healing wounds are a major concern in clinical practice and these wounds are mostly associated with diabetes, and venous and pressure ulcers. Wound healing is a complex process involving overlapping phases and the primary phase in this complex cascade is the inflammatory state. While inflammation is necessary for wound healing, a prolonged inflammatory phase leads to impaired healing. Cold-inducible RNA-binding protein (CIRP) belongs to a family of cold-shock proteins that are expressed in high levels under stress conditions. Recently, we demonstrated that a deficiency in CIRP led to decreased inflammation and mortality in an experimental model of hemorrhagic shock. Thus, we hypothesized that a deficiency in CIRP would accelerate the inflammatory phase and lead to an improvement in cutaneous wound healing. In this study, to examine this hypothesis, a full-thickness wound was created on the dorsum of wild-type (WT) and CIRP-/- mice. The wound size was measured every other day for 14 days. The wound area was significantly decreased in the CIRP-/- mice by day 9 and continued to decrease until day 14 compared to the WT mice. In a separate cohort, mice were sacrificed on days 3 and 7 after wounding and the skin tissues were harvested for histological analysis and RNA measurements. On day 3, the mRNA expression of tumor necrossis factor (TNF)-α in the skin tissues was increased by 16-fold in the WT mice, whereas these levels were increased by 65-fold in the CIRP-/- mice. Of note on day 7, while the levels of TNF-α remained high in the WT mice, these levels were significantly decreased in the CIRP-/- mice. The histological analysis of the wounded skin tissue indicated an improvement as early as day 3 in the CIRP-/- mice, whereas in the WT mice, infiltrated immune cells were still present on day 7. On day 7 in the CIRP-/- mice, Gr-1 expression was low and CD31 expression was high, whereas in the WT mice, Gr-1 expression was high and CD31 expression was low

  6. The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

    Directory of Open Access Journals (Sweden)

    Tam Michael WC

    2010-03-01

    Full Text Available Abstract Background Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. Results The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1 RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. Conclusions The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to

  7. Altered distributions of bone tissue mineral and collagen properties in women with fragility fractures.

    Science.gov (United States)

    Wang, Zhen Xiang; Lloyd, Ashley A; Burket, Jayme C; Gourion-Arsiquaud, Samuel; Donnelly, Eve

    2016-03-01

    Heterogeneity of bone tissue properties is emerging as a potential indicator of altered bone quality in pathologic tissue. The objective of this study was to compare the distributions of tissue properties in women with and without histories of fragility fractures using Fourier transform infrared (FTIR) imaging. We extended a prior study that examined the relationship of the mean FTIR properties to fracture risk by analyzing in detail the widths and the tails of the distributions of FTIR properties in biopsies from fracture and non-fracture cohorts. The mineral and matrix properties of cortical and trabecular iliac crest tissue were compared in biopsies from women with a history of fragility fracture (+Fx; n=21, age: mean 54±SD 15y) and with no history of fragility fracture (-Fx; n=12, age: 57±5y). A subset of the patients included in the -Fx group were taking estrogen-plus-progestin hormone replacement therapy (HRT) (-Fx+HRT n=8, age: 58±5y) and were analyzed separately from patients with no history of HRT (-Fx-HRT n=4, age: 56±7y). When the FTIR parameter mean values were examined by treatment group, the trabecular tissue of -Fx-HRT patients had a lower mineral:matrix ratio (M:M) and collagen maturity (XLR) than that of -Fx+HRT patients (-22% M:M, -18% XLR) and +Fx patients (-17% M:M, -18% XLR). Across multiple FTIR parameters, tissue from the -Fx-HRT group had smaller low-tail (5th percentile) values than that from the -Fx+HRT or +Fx groups. In trabecular collagen maturity and crystallinity (XST), the -Fx-HRT group had smaller low-tail values than those in the -Fx+HRT group (-16% XLR, -5% XST) and the +Fx group (-17% XLR, -7% XST). The relatively low values of trabecular mineral:matrix ratio and collagen maturity and smaller low-tail values of collagen maturity and crystallinity observed in the -Fx-HRT group are characteristic of younger tissue. Taken together, our data suggest that the presence of newly formed tissue that includes small/imperfect crystals

  8. Functional properties of butter oil made from bovine milk with experimentally altered fat composition.

    Science.gov (United States)

    Ortiz-Gonzalez, G; Jimenez-Flores, R; Bremmer, D R; Clark, J H; DePeters, E J; Schmidt, S J; Drackley, J K

    2007-11-01

    Modification of milk fat composition might be desirable to alter manufacturing characteristics or produce low saturated fat dairy products that more closely meet consumer dietary preferences. The aim of this research was to evaluate functional properties of butter oil obtained from milks with fat composition modified by altering the profile of long-chain fatty acids (FA) absorbed from the small intestine of cows. A control and 5 mixtures of long-chain free FA were infused into the abomasum of lactating dairy cows in a 6 x 6 Latin square design with 21-d periods. Treatments were 1) control (no FA infused), 2) mostly saturated FA (C16:C18 = 0.72), 3) low-linoleic palm FA (C16:C18 = 0.85), 4) palm FA (C16:C18 = 0.72), 5) soy FA (C16:C18 = 0.10), and 6) high-palmitic soy FA (C16:C18 = 0.68). All treatments included meat solubles and Tween 80 as emulsifiers. Solid fat content (from 0 to 40 degrees C), melting point, and force at fracture were determined in butter oil. Milk fat from cows infused with palm FA (treatment 4) exhibited functionality equal to or better than control butter oil. Infusion with palm FA increased amounts of triglyceride (TG) fractions with 48, 52, and 54 carbon numbers but decreased TG with 32, 34, 36, and 42 carbon numbers. Infusion with soy FA increased TG with 26, 38, 40, 52, and 54 carbon numbers but decreased TG with 34, 42, and 46 carbons. Infusion of the mostly saturated FA increased TG with 38, 50, 52, and 54 carbon numbers but decreased TG with 32, 34, and 42 carbon numbers. These TG groups were consistently correlated with functional properties of butter oils from different treatments. The content of palmitic acid is important for maintaining functionality in the presence of increased polyunsaturated FA. The composition of milk fat may be able to be optimized through nutritional manipulation of diets for dairy cows if the optimal composition of FA and TG is defined for a particular dairy product.

  9. Alterations of hydraulic soil properties influenced by land-use changes and agricultural management systems

    Science.gov (United States)

    Weninger, Thomas; Kreiselmeier, Janis; Chandrasekhar, Parvathy; Jülich, Stefan; Schwärzel, Kai; Schwen, Andreas

    2016-04-01

    Estimation and modeling of soil water movement and the hydrologic balance of soils requires sound knowledge about hydraulic soil properties (HSP). The soil water characteristics, the hydraulic conductivity function and the pore size distribution (PSD) are commonly used instruments for the mathematical representation of HSP. Recent research highlighted the temporal variability of these functions caused by meteorological or land-use influences. State of the art modeling software for the continuous simulation of soil water movement uses a stationary approach for the HSP which means that their time dependent alterations and the subsequent effects on soil water balance is not considered. Mathematical approaches to describe the evolution of PSD are nevertheless known, but there is a lack of sound data basis for parameter estimation. Based on extensive field and laboratory measurements at 5 locations along a climatic gradient across Austria and Germany, this study will quantify short-term changes in HSP, detect driving forces and introduce a method to predict the effects of soil and land management actions on the soil water balance. Amongst several soil properties, field-saturated and unsaturated hydraulic conductivities will be determined using a hood infiltration experiments in the field as well as by evaporation and dewpoint potentiometer method in the lab. All measurements will be carried out multiple times over a span of 2 years which will allow a detailed monitoring of changes in HSP. Experimental sites where we expect significant inter-seasonal changes will be equipped with sensors for soil moisture and matric potential. The choice of experimental field sites follows the intention to involve especially the effects of tillage operations, different cultivation strategies, microclimatically effective structures and land-use changes. The international project enables the coverage of a broad range of soil types as well as climate conditions and hence will have broad

  10. Alteration in cell surface properties of Burkholderia spp. during surfactant-aided biodegradation of petroleum hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, Sagarika; Mukherji, Suparna [Indian Institute of Technology Bombay, Mumbai (India). Centre for Environmental Science and Engineering (CESE)

    2012-04-15

    Chemical surfactants may impact microbial cell surface properties, i.e., cell surface hydrophobicity (CSH) and cell surface charge, and may thus affect the uptake of components from non-aqueous phase liquids (NAPLs). This work explored the impact of Triton X-100, Igepal CA 630, and Tween 80 (at twice the critical micelle concentration, CMC) on the cell surface characteristics of Burkholderia cultures, Burkholderia cepacia (ES1, aliphatic degrader) and Burkholderia multivorans (NG1, aromatic degrader), when grown on a six-component model NAPL. In the presence of Triton X-100, NAPL biodegradation was enhanced from 21% to 60% in B. cepacia and from 18% to 53% in B. multivorans. CSH based on water contact angle (50-52 ) was in the same range for both strains while zeta potential at neutral pH was -38 and -31 mV for B. cepacia and B. multivorans, respectively. In the presence of Triton X-100, their CSH increased to greater than 75 and the zeta potential decreased. This induced a change in the mode of uptake and initiated aliphatic hydrocarbon degradation by B. multivorans and increased the rate of aliphatic hydrocarbon degradation in B. cepacia. Igepal CA 630 and Tween 80 also altered the cell surface properties. For B. cepacia grown in the presence of Triton X-100 at two and five times its CMC, CSH increased significantly in the log growth phase. Growth in the presence of the chemical surfactants also affected the abundance of chemical functional groups on the cell surface. Cell surface changes had maximum impact on NAPL degradation in the presence of emulsifying surfactants, Triton X-100 and Igepal CA630.

  11. High temperatures alter physiological properties of pyramidal cells and inhibitory interneurons in hippocampus

    Directory of Open Access Journals (Sweden)

    Jennifer eKim

    2012-07-01

    Full Text Available Temperature has multiple effects on neurons, yet little is known about the effects of high temperature on the physiology of mammalian central neurons. Hyperthermia can influence behavior and cause febrile seizures. We studied the effects of acute hyperthermia on the immature hippocampus in vitro by recording from pyramidal neurons and inhibitory oriens-lacunosum moleculare (O-LM interneurons (identified by green fluorescent protein expression in the GIN mouse line. Warming to 41°C caused depolarization, spontaneous action potentials, reduced input resistance and membrane time constant, and increased spontaneous synaptic activity of most pyramidal cells and O-LM interneurons. Pyramidal neurons of area CA3 were more strongly excited by hyperthermia than those of area CA1. About 90% of O-LM interneurons in both CA1 and CA3 increased their firing rates at hyperthermic temperatures; interneurons in CA3 fired faster than those in CA1 on average. Blockade of fast synaptic transmission did not abolish the effect of hyperthermia on neuronal excitability. Our results suggest that hyperthermia increases hippocampal excitability, particularly in seizure-prone area CA3, by altering the intrinsic membrane properties of pyramidal cells and interneurons.

  12. High temperatures alter physiological properties of pyramidal cells and inhibitory interneurons in hippocampus.

    Science.gov (United States)

    Kim, Jennifer A; Connors, Barry W

    2012-01-01

    Temperature has multiple effects on neurons, yet little is known about the effects of high temperature on the physiology of mammalian central neurons. Hyperthermia can influence behavior and cause febrile seizures. We studied the effects of acute hyperthermia on the immature hippocampus in vitro by recording from pyramidal neurons and inhibitory oriens-lacunosum moleculare (O-LM) interneurons (identified by green fluorescent protein (GFP) expression in the GIN mouse line). Warming to 41°C caused depolarization, spontaneous action potentials, reduced input resistance and membrane time constant, and increased spontaneous synaptic activity of most pyramidal cells and O-LM interneurons. Pyramidal neurons of area CA3 were more strongly excited by hyperthermia than those of area CA1. About 90% of O-LM interneurons in both CA1 and CA3 increased their firing rates at hyperthermic temperatures; interneurons in CA3 fired faster than those in CA1 on average. Blockade of fast synaptic transmission did not abolish the effect of hyperthermia on neuronal excitability. Our results suggest that hyperthermia increases hippocampal excitability, particularly in seizure-prone area CA3, by altering the intrinsic membrane properties of pyramidal cells and interneurons.

  13. Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

    Science.gov (United States)

    Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

    In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

  14. Influence of diet-mediated maternal thyroid alterations on functional properties of turkey eggs.

    Science.gov (United States)

    Christensen, V L; Ort, J F

    1990-09-01

    Maternal thyroid status was altered by means of diet to determine its effect on functional properties of turkey eggshells. Hens were fed a control diet (CON), the CON diet containing .5 ppm triiodothyronine (T3), the CON diet containing 2.1 ppm iodine as potassium iodide (KI) or the CON diet containing .1% thiouracil (THIO). Feeding T3 decreased plasma thyroxine but elevated plasma T3 concentrations compared to CON. The KI diet had no effect on plasma thyroid hormone concentrations, but feeding THIO depressed plasma thyroxine with no effect on T3, resulting in an elevated ratio of the two hormones compared to the CON ratio. Feeding KI decreased egg volume and T3 increased egg density compared to CON, but no effects on egg weight, surface area, width, or length were noted. Dietary T3 depressed eggshell water vapor conductance compared to CON. Dietary iodine resulted in thinner eggshells with fewer pores than the CON, whereas THIO caused significantly more pores in eggshells than CON but had no effects on shell thickness. Dietary KI had no effects on maternal plasma thyroid hormone concentrations, suggesting that the effects were due to iodine availability rather than to thyroid hormones. It is concluded that the availability of iodine to turkey breeder hens may influence eggshell characteristics.

  15. Deformability in the cleavage site of primary microRNA is not sensed by the double-stranded RNA binding domains in the microprocessor component DGCR8.

    Science.gov (United States)

    Quarles, Kaycee A; Chadalavada, Durga; Showalter, Scott A

    2015-06-01

    The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency.

  16. Developmental expression of Musashi-1 and Musashi-2 RNA-binding proteins during spermatogenesis: analysis of the deleterious effects of dysregulated expression.

    Science.gov (United States)

    Sutherland, Jessie M; Fraser, Barbara A; Sobinoff, Alexander P; Pye, Victoria J; Davidson, Tara-Lynne; Siddall, Nicole A; Koopman, Peter; Hime, Gary R; McLaughlin, Eileen A

    2014-05-01

    Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development.

  17. The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins.

    Science.gov (United States)

    Dasgupta, Twishasri; Ladd, Andrea N

    2012-01-01

    RNA processing is important for generating protein diversity and modulating levels of protein expression. The CUG-BP, Elav-like family (CELF) of RNA-binding proteins regulate several steps of RNA processing in the nucleus and cytoplasm, including pre-mRNA alternative splicing, C to U RNA editing, deadenylation, mRNA decay, and translation. In vivo, CELF proteins have been shown to play roles in gametogenesis and early embryonic development, heart and skeletal muscle function, and neurosynaptic transmission. Dysregulation of CELF-mediated programs has been implicated in the pathogenesis of human diseases affecting the heart, skeletal muscles, and nervous system.

  18. Altered synaptic properties during integration of adult-born hippocampal neurons following a seizure insult.

    Directory of Open Access Journals (Sweden)

    Johanna Jackson

    Full Text Available Pathological conditions affect several stages of neurogenesis in the adult brain, including proliferation, survival, cell fate, migration, and functional integration. Here we explored how a pathological environment modulates the heterogeneous afferent synaptic input that shapes the functional properties of newly formed neurons. We analyzed the expression of adhesion molecules and other synaptic proteins on adult-born hippocampal neurons formed after electrically-induced partial status epilepticus (pSE. New cells were labeled with a GFP-retroviral vector one week after pSE. One and three weeks thereafter, synaptic proteins were present on dendritic spines and shafts, but without differences between pSE and control group. In contrast, at six weeks, we found fewer dendritic spines and decreased expression of the scaffolding protein PSD-95 on spines, without changes in expression of the adhesion molecules N-cadherin or neuroligin-1, primarily located at excitatory synapses. Moreover, we detected an increased expression of the inhibitory scaffolding protein gephyrin in newborn but not mature neurons after SE. However, this increase was not accompanied by a difference in GABA expression, and there was even a region-specific decrease in the adhesion molecule neuroligin-2 expression, both in newborn and mature neurons. Neuroligin-2 clusters co-localized with presynaptic cholecystokinin terminals, which were also reduced. The expression of neuroligin-4 and glycine receptor was unchanged. Increased postsynaptic clustering of gephyrin, without an accompanying increase in GABAergic input or neuroligin-2 and -4 expression, the latter important for clustering of GABA(A and glycine receptors, respectively, could imply an increased but altered inhibitory connectivity specific for newborn neurons. The changes were transient and expression of both gephyrin and NL-2 was normalized 3 months post-SE. Our findings indicate that seizure-induced brain pathology alters

  19. Acid weathering of basalt and basaltic glass: 2. Effects of microscopic alteration textures on spectral properties

    Science.gov (United States)

    Smith, Rebecca J.; Horgan, Briony H. N.; Mann, Paul; Cloutis, Edward A.; Christensen, Philip R.

    2017-01-01

    Acid alteration has long been proposed for the Martian surface, and so it is important to understand how the resulting alteration textures affect surface spectra. Two basaltic materials of varying crystallinity were altered in two different H2SO4 solutions (pH 1 and pH 3) for 220 days. The unaltered and altered samples were studied in the visible and near infrared (VNIR) and thermal infrared (TIR), and select samples were chosen for scanning electron microscopy analysis. Materials altered in pH 3 solutions showed little to no physical alteration, and their spectral signatures changed very little. In contrast, all materials altered in pH 1 acid displayed silica-rich alteration textures, and the morphology differed based on starting material crystallinity. The more crystalline material displayed extensive alteration reaching into the sample interiors and had weaker silica spectral features. The glass sample developed alteration layers tens of microns thick, exhibiting amorphous silica-rich spectral features that completely obscured the substrate. Thus, the strong absorption coefficient of silica effectively decreases the penetration depth of TIR spectral measurements, causing silica abundances to be grossly overestimated in remote sensing data. Additionally, glass samples with silica layers exhibited distinct concave up blue spectral slopes in the VNIR. Spectra from the northern lowland plains of Mars are modeled with high abundances of amorphous silica and exhibit concave up blue spectral slopes and are thus consistent with acid altered basaltic glass. Therefore, we conclude that large regions of the Martian surface may have formed through the interaction of basaltic glass with strongly acidic fluids.

  20. Seismic properties and effects of hydrothermal alteration on Volcanogenic Massive Sulfide (VMS) deposits at the Lalor Lake in Manitoba, Canada

    Science.gov (United States)

    Miah, Khalid H.; Bellefleur, Gilles; Schetselaar, Ernst; Potter, David K.

    2015-12-01

    Borehole sonic and density logs are essential for mineral exploration at depth, but its limited availability to link rock properties of different ore forming geologic structure is a hindrance to seismic data interpretations. In situ density and velocity logs provide first order control on the reflectivity of various lithologic units. We analyzed borehole logs from 12 drill holes over and around the Lalor VMS deposits geographically located in the northern Manitoba, Canada, in an attempt to characterize lithologic units based on its seismic properties. The Lalor Lake deposit is part of the Paleoproterozoic Flin Flon Belt, and associated with an extensive hydrothermal alteration system. Volcanogenic Massive Sulfide (VMS) zones are distributed in several ore lenses with relatively shallower facies comprise solid to solid sulfides, tend to be disseminated or Stringer sulfides, while deeper lenses are gold and silver enriched and occurred in the highly altered footwall region. Our analysis suggests that massive sulfide and diorite have higher acoustic impedance than other rock units, and can produce useful reflection signatures in seismic data. Bivariate distributions of P-wave velocity, density, acoustic impedance and Poisson's ratio in end-member mineral cones were used for qualitative assessment of the extent of alteration of various lithologic units. It can be inferred that hydrothermal alteration has considerably increased P-wave velocity and density of altered argillite and felsic volcanic rocks in comparison to their corresponding unaltered facies. Amphibole, garnet, kyanite, pyrite, sphalerite and staurolite are the dominant end-member alteration minerals affecting seismic rock properties at the VMS site.

  1. ALTERATIONS OF PROPERTIES OF RED BLOOD CELLS MEMBRANES PROTEINS OF DIFFERENT AGE AND SEX VOLUNTEERS.

    Science.gov (United States)

    Pruidze, N; Khetsuriani, R; Sujashvili, R; Ioramashvili, I; Arabuli, M; Sanikidze, T

    2015-01-01

    disorders of protein-protein interaction mechanisms, their ubiquitinylation or oligomerisation and formation of high molecular weight complexes of inactivated proteins in aged RBCs. These processes play important role in regulation of the RBCs shape and stability. Identified sex- and age-related alterations in RBCs membranes proteins affect the rheological properties of blood and can be considered as the etiologic and pathogenic markers of various diseases.

  2. A KH Domain-Containing Putative RNA-Binding Protein Is Critical for Heat Stress-Responsive Gene Regulation and Thermotolerance in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Qingmei Guan; Changlong Wen; Haitao Zeng; Jianhua Zhu

    2013-01-01

    Heat stress is a severe environmental factor that significantly reduces plant growth and delays development.Heat stress factors (HSFs) are a class of transcription factors that are synthesized rapidly in response to elevations in temperature and are responsible for the transcription of many heat stress-responsive genes including those encoding heat shock proteins (HSPs).There are 21 HSFs in Arabidopsis,and recent studies have established that the HSFA1 family members are master regulators for the remaining HSFs.However,very little is known about upstream molecular factors that control the expression of HSFA1 genes and other HSF genes under heat stress.Through a forward genetic analysis,we identified RCF3,a K homology (KH) domain-containing nuclear-localized putative RNA-binding protein.RCF3 is a negative regulator of most HSFs,including HSFAla,HSFAlb,and HSFAld.In contrast,RCF3 positively controls the expression of HSFAle,HSFA3,HSFA9,HSFB3,and DREB2C.Consistently with the overall increased accumulation of heat-responsive genes,the rcf3 mutant plants are more tolerant than the wild-type to heat stress.Together,our results suggest that a KH domain-containing putative RNA-binding protein RCF3 is an important upstream regulator for heat stress-responsive gene expression and thermotolerance in Arabidopsis.

  3. Associations of homologous RNA-binding motif gene on the X chromosome (RBMX) and its like sequence on chromosome 9(RBMXL9) with non-obstructive azoospermia

    Institute of Scientific and Technical Information of China (English)

    Akira Tsujimura; Masao Ota; Akihiko Okuyama; Kazutoshi Fujita; Kazuhiko Komori; Phanu Tanjapatkul; Yasushi Miyagawa; Shingo Takada; Kiyomi Matsumiya; Masaharu Sada; Yoshihiko Katsuyama

    2006-01-01

    Aim: To investigate the associations of autosomal and X-chromosome homologs of the RNA-binding-motif (RNA-binding-motif on the Y chromosome, RBMY) gene with non-obstructive azoospermia (NOA), as genetic factors for NOA may map to chromosomes other than the Y chromosome. Methods: Genomic DNA was extracted using a salting-out procedure after treatment of peripheral blood leukocytes with proteinase K from Japanese patients with NOA (n = 67) and normal fertile volunteers (n = 105). The DNA were analyzed for RBMX by expressed sequence tag (EST) deletion and for the like sequence on chromosome 9 (RBMXL9) by microsatellite polymorphism. Results: We examined six ESTs in and around RBMX and found a deletion of SHGC31764 in one patient with NOA and a deletion of DXS7491 in one other patient with NOA. No deletions were detected in control subjects. The association study with nine microsatellite markers near RBMXL9 revealed that D9S319 was less prevalent in patients than in control subjects, whereas D9S1853 was detected more frequently in patients than that in control subjects. Conclusion: We provide evidence that deletions in or around RBMX may be involved in NOA. In addition, analyses of markers in the vicinity of RBMXL9 on chromosome 9 suggest the possibility that variants of this gene may be associated with NOA.Although further studies are necessary, this is the first report of the association between RBMX and RBMXL9 with NOA.

  4. Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

    Energy Technology Data Exchange (ETDEWEB)

    Weidmann, Chase A.; Qiu, Chen; Arvola, René M.; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T.; Tanaka Hall, Traci M.; Goldstrohm, Aaron C.

    2016-08-02

    Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation byDrosophilaPumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulatedin vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

  5. A cDNA encoding a cold-induced glycine-rich RNA binding protein from Prunus avium expressed in embryonic axes.

    Science.gov (United States)

    Stephen, John R; Dent, Katherine C; Finch-Savage, William E

    2003-11-27

    A cDNA clone encoding a presumed full-length glycine-rich ribonucleic acid (RNA) binding protein was isolated from a lambda-ZAP Express cDNA library generated from primarily nondormant Prunus avium (wild cherry) embryonic axes. The cDNA, designated Pa-RRM-GRP1 (Prunus avium RNA recognition motif glycine-rich protein 1), contains a single N-terminal RNA recognition motif (RRM) and single C-terminal glycine-rich domain. The glycine-rich domain is unusually long at 91 amino acids, 58 of which are glycines. The 534-base pair (bp) open reading frame (ORF) of this clone encodes a 178-amino-acid polypeptide with a predicted molecular weight of 17.33 kDa and pI of 7.84. Comparative sequence alignment of Pa-RRM-GRP1 reveals extensive homology to known and presumed glycine-rich RNA binding proteins from angiosperms and gymnosperms. Genomic Southern blot analysis suggests that this gene exists as a single copy in P. avium. Expression of this gene in P. avium embryonic axes during low-temperature dormancy-breaking treatments was studied and found to be induced by cold (3 degrees C) using real-time PCR of total cDNA supported by Northern blot analysis of total RNA. Expression dropped during prolonged storage at 3 degrees C and was reduced to control levels by interruption of cold treatment by warming to 20 degrees C.

  6. Evolutionary history of double-stranded RNA binding proteins in plants: identification of new cofactors involved in easiRNA biogenesis.

    Science.gov (United States)

    Clavel, Marion; Pélissier, Thierry; Montavon, Thomas; Tschopp, Marie-Aude; Pouch-Pélissier, Marie-Noëlle; Descombin, Julie; Jean, Viviane; Dunoyer, Patrice; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc

    2016-05-01

    In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

  7. Two basic (hydrophilic) regions in the movement protein of Parietaria mottle virus have RNA binding activity and are required for cell-to-cell transport.

    Science.gov (United States)

    Martínez, Carolina; Coll-Bonfill, Nuria; Aramburu, Jose; Pallás, Vicente; Aparicio, Frederic; Galipienso, Luis

    2014-05-12

    The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell movement. Bioinformatics analysis identified two hydrophilic non-contiguous regions (R1 and R2) rich in the basic amino acids lysine and arginine and with the predicted secondary structure of an α-helix. Different approaches were used to determine the implication of the R1 and R2 regions in RNA binding, plasmodesmata (PD) targeting and cell-to-cell movement. EMSA (Electrophoretic Mobility Shift Assay) showed that both regions have RNA-binding activity whereas that mutational analysis reported that either deletion of any of these regions, or loss of the basic amino acids, interfered with the viral intercellular movement. Subcellular localization studies showed that PMoV MP locates at PD. Mutants designed to impeded cell-to-cell movement failed to accumulate at PD indicating that basic residues in both R1 and R2 are critical for binding the MP at PD.

  8. Long Non-Coding RNA HOTAIR Promotes Cell Migration and Invasion via Down-Regulation of RNA Binding Motif Protein 38 in Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Chaofeng Ding

    2014-03-01

    Full Text Available Long non-coding RNA HOTAIR exerts regulatory functions in various biological processes in cancer cells, such as proliferation, apoptosis, mobility, and invasion. We previously found that HOX transcript antisense RNA (HOTAIR is a negative prognostic factor and exhibits oncogenic activity in hepatocellular carcinoma (HCC. In this study, we aimed to investigate the role and molecular mechanism of HOTAIR in promoting HCC cell migration and invasion. Firstly, we profiled its gene expression pattern by microarray analysis of HOTAIR loss in Bel-7402 HCC cell line. The results showed that 129 genes were significantly down-regulated, while 167 genes were significantly up-regulated (fold change >2, p < 0.05. Bioinformatics analysis indicated that RNA binding proteins were involved in this biological process. HOTAIR suppression using RNAi strategy with HepG2 and Bel-7402 cells increased the mRNA and protein expression levels of RNA binding motif protein 38 (RBM38. Moreover, the expression levels of RBM38 in HCC specimens were significantly lower than paired adjacent noncancerous tissues. In addition, knockdown of HOTAIR resulted in a decrease of cell migration and invasion, which could be specifically rescued by down-regulation of RBM38. Taken together, HOTAIR could promote migration and invasion of HCC cells by inhibiting RBM38, which indicated critical roles of HOTAIR and RBM38 in HCC progression.

  9. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation.

    Science.gov (United States)

    Chang, Xing; Li, Bin; Rao, Anjana

    2015-04-14

    Posttranscriptional regulation is a major mechanism to rewire transcriptomes during differentiation. Heterogeneous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including effector T cells and plasma cells. To study the molecular functions of hnRNPLL at a genome-wide level, we identified hnRNPLL RNA targets and binding sites in plasma cells through integrated Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) and RNA sequencing. hnRNPLL preferentially recognizes CA dinucleotide-containing sequences in introns and 3' untranslated regions (UTRs), promotes exon inclusion or exclusion in a context-dependent manner, and stabilizes mRNA when associated with 3' UTRs. During differentiation of primary B cells to plasma cells, hnRNPLL mediates a genome-wide switch of RNA processing, resulting in loss of B-cell lymphoma 6 (Bcl6) expression and increased Ig production--both hallmarks of plasma-cell maturation. Our data identify previously unknown functions of hnRNPLL in B-cell to plasma-cell differentiation and demonstrate that the RNA-binding protein hnRNPLL has a critical role in tuning transcriptomes of terminally differentiating B lymphocytes.

  10. The Zea mays glycine-rich RNA-binding protein MA16 is bound to a ribonucleotide(s) by a stable linkage.

    Science.gov (United States)

    Freire, Miguel Angel

    2012-09-01

    Expression of the gene encoding the maize glycine-rich RNA-binding protein MA16 is developmentally regulated and it is involved in environmental stress responses. The MA16 protein shows a wide spectrum of RNA-binding activities. On the basis of in vivo labelling, where a [³²P]phosphate label was linked to the MA16 protein, Freire and Pages (Plant Mol Biol 29:797-807, 1995) suggested that the protein may be post-translationally modified by phosphorylation. However, further analysis showed that the [³²P]phosphate label was sensitive to different treatments, suggesting that modification distinct from protein phosphorylation might occur in the MA16 protein. Biochemical analysis revealed that this [³²P]phosphate labelling was resistant to phenol extraction and denaturing SDS-PAGE but sensitive to micrococcal nuclease, RNase A and RNase T1 treatments. The mobility of [³⁵S] labelled MA16 protein on SDS-PAGE did not significantly changed after the nuclease treatments suggesting that the [³²P]phosphate label associated to MA16 protein could be a ribonucleotide or a very short ribonucleotide chain. In addition, immunoprecipitation of labelled extracts showed that the ribonucleotide(s) linked to the MA16 protein was removed by phosphorolytic activity. This activity could be catalysed by a phosphate-dependent ribonuclease. The C-terminus of MA16 protein harbouring a glycine-rich domain was predicted to be an intrinsically disordered region.

  11. A non-proteolytic role for ubiquitin in deadenylation of MHC-I mRNA by the RNA-binding E3-ligase MEX-3C

    Science.gov (United States)

    Cano, Florencia; Rapiteanu, Radu; Sebastiaan Winkler, G.; Lehner, Paul J.

    2015-01-01

    The regulation of protein and mRNA turnover is essential for many cellular processes. We recently showed that ubiquitin—traditionally linked to protein degradation—directly regulates the degradation of mRNAs through the action of a newly identified family of RNA-binding E3 ubiquitin ligases. How ubiquitin regulates mRNA decay remains unclear. Here, we identify a new role for ubiquitin in regulating deadenylation, the initial and often rate-limiting step in mRNA degradation. MEX-3C, a canonical member of this family of RNA-binding ubiquitin ligases, associates with the cytoplasmic deadenylation complexes and ubiquitinates CNOT7(Caf1), the main catalytic subunit of the CCR4-NOT deadenylation machinery. We establish a new role for ubiquitin in regulating MHC-I mRNA deadenylation as ubiquitination of CNOT7 by MEX-3C regulates its deadenylation activity and is required for MHC-I mRNA degradation. Since neither proteasome nor lysosome inhibitors rescued MEX-3C-mediated MHC-I mRNA degradation, our findings suggest a new non-proteolytic function for ubiquitin in the regulation of mRNA decay. PMID:26471122

  12. Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

    Science.gov (United States)

    Samatanga, Brighton; Andreou, Alexandra Z; Klostermeier, Dagmar

    2017-01-23

    DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

  13. A functional SNP catalog of overlapping miRNA-binding sites in genes implicated in prion disease and other neurodegenerative disorders.

    Science.gov (United States)

    Saba, Reuben; Medina, Sarah J; Booth, Stephanie A

    2014-10-01

    The involvement of SNPs in miRNA target sites remains poorly investigated in neurodegenerative disease. In addition to associations with disease risk, such genetic variations can also provide novel insight into mechanistic pathways that may be responsible for disease etiology and/or pathobiology. To identify SNPs associated specifically with degenerating neurons, we restricted our analysis to genes that are dysregulated in CA1 hippocampal neurons of mice during early, preclinical phase of Prion disease. The 125 genes chosen are also implicated in other numerous degenerative and neurological diseases and disorders and are therefore likely to be of fundamental importance. We predicted those SNPs that could increase, decrease, or have neutral effects on miRNA binding. This group of genes was more likely to possess DNA variants than were genes chosen at random. Furthermore, many of the SNPs are common within the human population, and could contribute to the growing awareness that miRNAs and associated SNPs could account for detrimental neurological states. Interestingly, SNPs that overlapped miRNA-binding sites in the 3'-UTR of GABA-receptor subunit coding genes were particularly enriched. Moreover, we demonstrated that SNP rs9291296 would strengthen miR-26a-5p binding to a highly conserved site in the 3'-UTR of gamma-aminobutyric acid receptor subunit alpha-4.

  14. Dynamic wettability alteration in immiscible two-phase flow in porous media: Effect on transport properties and critical slowing down

    Directory of Open Access Journals (Sweden)

    Vegard eFlovik

    2015-11-01

    Full Text Available The change in contact angles due to the injection of low salinity water or any other wettability altering agent in an oil-rich porous medium is modeled by a network model of disordered pores transporting two immiscible fluids. We introduce a dynamic wettability altering mechanism, where the time dependent wetting property of each pore is determined by the cumulative flow of water through it. Simulations are performed to reach steady-state for different possible alterations in the wetting angle (θ. We find that deviation from oil-wet conditions re-mobilizes the stuck clusters and increases the oil fractional flow. However, the rate of increase in the fractional flow depends strongly on θ and as θ → 90◦ , a critical angle, the system shows critical slowing down which is characterized by two dynamic critical exponents.

  15. Dynamic wettability alteration in immiscible two-phase flow in porous media: Effect on transport properties and critical slowing down

    CERN Document Server

    Flovik, Vegard; Hansen, Alex

    2015-01-01

    The change in contact angles due to the injection of low salinity water or any other wettability altering agent in an oil-rich porous medium is modeled by a network model of disordered pores transporting two immiscible fluids. We introduce a dynamic wettability altering mechanism, where the time dependent wetting property of each pore is determined by the cumulative flow of water through it. Simulations are performed to reach steady-state for different possible alterations in the wetting angle ($\\theta$). We find that deviation from oil-wet conditions re-mobilizes the stuck clusters and increases the oil fractional flow. However, the rate of increase in the fractional flow depends strongly on $\\theta$ and as $\\theta\\to 90^\\circ$, a critical angle, the system shows critical slowing down which is characterized by two dynamic critical exponents.

  16. Altered topological properties of functional network connectivity in schizophrenia during resting state: a small-world brain network study.

    Science.gov (United States)

    Yu, Qingbao; Sui, Jing; Rachakonda, Srinivas; He, Hao; Gruner, William; Pearlson, Godfrey; Kiehl, Kent A; Calhoun, Vince D

    2011-01-01

    Aberrant topological properties of small-world human brain networks in patients with schizophrenia (SZ) have been documented in previous neuroimaging studies. Aberrant functional network connectivity (FNC, temporal relationships among independent component time courses) has also been found in SZ by a previous resting state functional magnetic resonance imaging (fMRI) study. However, no study has yet determined if topological properties of FNC are also altered in SZ. In this study, small-world network metrics of FNC during the resting state were examined in both healthy controls (HCs) and SZ subjects. FMRI data were obtained from 19 HCs and 19 SZ. Brain images were decomposed into independent components (ICs) by group independent component analysis (ICA). FNC maps were constructed via a partial correlation analysis of ICA time courses. A set of undirected graphs were built by thresholding the FNC maps and the small-world network metrics of these maps were evaluated. Our results demonstrated significantly altered topological properties of FNC in SZ relative to controls. In addition, topological measures of many ICs involving frontal, parietal, occipital and cerebellar areas were altered in SZ relative to controls. Specifically, topological measures of whole network and specific components in SZ were correlated with scores on the negative symptom scale of the Positive and Negative Symptom Scale (PANSS). These findings suggest that aberrant architecture of small-world brain topology in SZ consists of ICA temporally coherent brain networks.

  17. Search for limiting factors in the RNAi pathway in silkmoth tissues and the Bm5 cell line: the RNA-binding proteins R2D2 and Translin.

    Science.gov (United States)

    Swevers, Luc; Liu, Jisheng; Huvenne, Hanneke; Smagghe, Guy

    2011-01-01

    RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

  18. Nitrogen deposition alters soil chemical properties and bacterial communities in the Inner Mongolia grassland

    Institute of Scientific and Technical Information of China (English)

    Ximei Zhang; Xingguo Han

    2012-01-01

    Nitrogen deposition has dramatically altered biodiversity and ecosystem functioning on the earth; however,its effects on soil bacterial community and the underlying mechanisms of these effects have not been thoroughly examined.Changes in ecosystems caused by nitrogen deposition have traditionally been attributed to increased nitrogen content.In fact,nitrogen deposition not only leads to increased soil total N content,but also changes in the NH4+-N content,NO3--N content and pH,as well as changes in the heterogeneity of the four indexes.The soil indexes for these four factors,their heterogeneity and even the plant community might be routes through which nitrogen deposition alters the bacterial community.Here,we describe a 6-year nitrogen addition experiment conducted in a typical steppe ecosystem to investigate the ecological mechanism by which nitrogen deposition alters bacterial abundance,diversity and composition.We found that various characteristics of the bacterial community were explained by different environmental factors.Nitrogen deposition decreased bacterial abundance that is positively related to soil pH value.In addition,nitrogen addition decreased bacterial diversity,which is negatively related to soil total N content and positively related to soil NO3--N heterogeneity.Finally,nitrogen.addition altered bacterial composition that is significantly related to soil NH4+-N content.Although nitrogen deposition significantly altered plant biomass,diversity and composition,these characteristics of plant community did not have a significant impact on processes of nitrogen deposition that led to alterations in bacterial abundance,diversity and composition.Therefore,more sensitive molecular technologies should be adopted to detect the subtle shifts of microbial community structure induced by the changes of plant community upon nitrogen deposition.

  19. NH125 reduces the level of CPEB3, an RNA binding protein, to promote synaptic GluA2 expression.

    Science.gov (United States)

    Bender, Crhistian L; Yang, Qian; Sun, Lu; Liu, Siqiong June

    2016-02-01

    Neuronal activity can alter the phosphorylation state of eukaryotic elongation factor 2 (eEF2) and thereby regulates protein synthesis. This is thought to be the underlying mechanism for a form of synaptic plasticity that involves changes in the expression of synaptic AMPA type glutamate receptors. Phosphorylation of eEF2 by Ca/calmodulin-dependent eEF2 kinase reduces the activity of eEF2, and this is prevented by a commonly used eEF2 kinase inhibitor, NH125. Here we show that 10 μM NH125 increased the expression of synaptic GluA2-containing receptors in mouse cerebellar stellate cells and this was prevented by a protein synthesis inhibitor. However NH125 at 10 μM also reduced the level of CPEB3, a protein that is known to bind to GluA2 mRNA and suppress GluA2 (also known as GluR2) synthesis. In contrast, a low concentration of NH125 lowered the peEF2 level, but did not alter CPEB3 expression and also failed to increase synaptic GluA2 receptors. A selective eEF2 kinase inhibitor, A-484954, decreased the level of peEF2, without changing the expression of CPEB3. This suggests that reducing peEF2 does not lead to a decrease in CPEB3 levels and is not sufficient to increase GluA2 synthesis. Thus NH125 at 10 μM reduced the level of CPEB3, and promoted GluA2 translation via a mechanism independent of inhibition of eEF2 kinase. Therefore NH125 does not always alter protein synthesis via selective inhibition of eEF2 kinase and the effects of NH125 on translation of mRNAs should be interpreted with caution.

  20. Discovery of the first small molecule inhibitor of human DDX3 specifically designed to target the RNA binding site: towards the next generation HIV-1 inhibitors.

    Science.gov (United States)

    Radi, Marco; Falchi, Federico; Garbelli, Anna; Samuele, Alberta; Bernardo, Vincenzo; Paolucci, Stefania; Baldanti, Fausto; Schenone, Silvia; Manetti, Fabrizio; Maga, Giovanni; Botta, Maurizio

    2012-03-01

    Efficacy of currently approved anti-HIV drugs is hampered by mutations of the viral enzymes, leading invariably to drug resistance and chemotherapy failure. Recent data suggest that cellular co-factors also represent useful targets for anti-HIV therapy. Here we describe the identification of the first small molecules specifically designed to inhibit the HIV-1 replication by targeting the RNA binding site of the human DEAD-Box RNA helicase DDX3. Optimization of a easily synthetically accessible hit (1) identified by application of a high-throughput docking approach afforded the promising compounds 6 and 8 which proved to inhibit both the helicase and ATPase activity of DDX3 and to reduce the viral load of peripheral blood mononuclear cells (PBMC) infected with HIV-1.

  1. The RNA-binding protein Xp54nrb isolated from a Ca²+-dependent screen is expressed in neural structures during Xenopus laevis development.

    Science.gov (United States)

    Neant, Isabelle; Deisig, Nina; Scerbo, Pierluigi; Leclerc, Catherine; Moreau, Marc

    2011-01-01

    In amphibian embryos, calcium (Ca(2+)) signalling is a necessary and sufficient event to induce neural fate. Transient elevations of [Ca(2+)]i are recorded in neural tissue precursor cells in whole embryos during gastrulation. Using a subtractive cDNA library between control ectoderm (animal caps) and ectoderm induced toward a neural fate by Ca(2+) release, we have isolated several Ca(2+)-induced target genes. Among the isolated genes, Xp54nrb encodes a protein which exhibits the RRM domains characteristic of RNA binding proteins, and is implicated in pre-mRNA splicing steps. Here we show that the Xp54nrb transcripts are expressed throughout early developmental stages, specifically in the neural and sensorial territories and that Xp54nrb could be involved in anterior neural patterning.

  2. Molecular cloning, expression pattern, and 3D structural prediction of the cold inducible RNA-binding protein (CIRP) in Japanese flounder ( Paralichthys olivaceus)

    Science.gov (United States)

    Yang, Xiao; Gao, Jinning; Ma, Liman; Li, Zan; Wang, Wenji; Wang, Zhongkai; Yu, Haiyang; Qi, Jie; Wang, Xubo; Wang, Zhigang; Zhang, Quanqi

    2015-02-01

    Cold-inducible RNA-binding protein (CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first cloned from mammals. On the contrary, there are little reports in teleosts. In this study, the Po CIRP gene of the Japanese flounder was cloned and sequenced. The genomic sequence consists of seven exons and six introns. The putative PoCIRP protein of flounder was 198 amino acid residues long containing the RNA recognition motif (RRM). Phylogenetic analysis showed that the flounder PoCIRP is highly conserved with other teleost CIRPs. The 5' flanking sequence was cloned by genome walking and many transcription factor binding sites were identified. There is a CpGs region located in promoter and exon I region and the methylation state is low. Quantitative real-time PCR analysis uncovered that Po CIRP gene was widely expressed in adult tissues with the highest expression level in the ovary. The mRNA of the Po CIRP was maternally deposited and the expression level of the gene was regulated up during the gastrula and neurula stages. In order to gain the information how the protein interacts with mRNA, we performed the modeling of the 3D structure of the flounder PoCIRP. The results showed a cleft existing the surface of the molecular. Taken together, the results indicate that the CIRP is a multifunctional molecular in teleosts and the findings about the structure provide valuable information for understanding the basis of this protein's function.

  3. Myxoma Virus dsRNA Binding Protein M029 Inhibits the Type I IFN-Induced Antiviral State in a Highly Species-Specific Fashion

    Science.gov (United States)

    Rahman, Masmudur M.; McFadden, Grant

    2017-01-01

    Myxoma virus (MYXV) is a Leporipoxvirus that possesses a specific rabbit-restricted host tropism but exhibits a much broader cellular host range in cultured cells. MYXV is able to efficiently block all aspects of the type I interferon (IFN)-induced antiviral state in rabbit cells, partially in human cells and very poorly in mouse cells. The mechanism(s) of this species-specific inhibition of type I IFN-induced antiviral state is not well understood. Here we demonstrate that MYXV encoded protein M029, a truncated relative of the vaccinia virus (VACV) E3 double-stranded RNA (dsRNA) binding protein that inhibits protein kinase R (PKR), can also antagonize the type I IFN-induced antiviral state in a highly species-specific manner. In cells pre-treated with type I IFN prior to infection, MYXV exploits M029 to overcome the induced antiviral state completely in rabbit cells, partially in human cells, but not at all in mouse cells. However, in cells pre-infected with MYXV, IFN-induced signaling is fully inhibited even in the absence of M029 in cells from all three species, suggesting that other MYXV protein(s) apart from M029 block IFN signaling in a species-independent manner. We also show that the antiviral state induced in rabbit, human or mouse cells by type I IFN can inhibit M029-knockout MYXV even when PKR is genetically knocked-out, suggesting that M029 targets other host proteins for this antiviral state inhibition. Thus, the MYXV dsRNA binding protein M029 not only antagonizes PKR from multiple species but also blocks the type I IFN antiviral state independently of PKR in a highly species-specific fashion. PMID:28157174

  4. Genetic variants within microRNA-binding site of RAD51B are associated with risk of cervical cancer in Chinese women.

    Science.gov (United States)

    Hang, Dong; Zhou, Wen; Jia, Meiqun; Wang, Lihua; Zhou, Jing; Yin, Yin; Ma, Hongxia; Hu, Zhibin; Li, Ni; Shen, Hongbin

    2016-09-01

    RAD51B plays a central role in homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs), which is important to prevent genomic instability, a hallmark of cancer. Recent studies suggested that common genetic variants of RAD51B may contribute to cancer susceptibility. In this study, we aimed to investigate whether potentially functional variants within miRNA-binding sites of RAD51B are associated with risk of cervical cancer. A total of 1486 cervical cancer patients and 1536 cancer-free controls were enrolled, and two genetic variants, rs963917 (A > G) and rs963918 (T > C), were genotyped in all participants. Using multivariate logistic regression analyses, we found that G allele of rs963917 conferred lower risk of cervical cancer compared to A allele (adjusted OR = 0.89, 95% CI = 0.80-0.99, P = 0.039). Similarly, rs963918 allele C was associated with a decreased risk for cervical cancer compared with allele T (adjusted OR = 0.84, 95% CI = 0.74-0.94, P = 0.004). Haplotype analyses showed that haplotype GC was also correlated with lower risk (OR = 0.83, 95% CI = 0.73-0.95, P = 0.005) compared with the most common haplotype AT. In summary, our study suggested that miRNA-binding site genetic variants of RAD51B may modify the susceptibility to cervical cancer, which is important to identify individuals with differential risk for this malignancy and to improve the effectiveness of preventive intervention.

  5. AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins.

    Science.gov (United States)

    Parida, Adwaita Prasad; Sharma, Amrapali; Sharma, Arun Kumar

    2017-03-01

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

  6. Molecular Cloning, Expression Pattern, and 3D Structural Prediction of the Cold Inducible RNA - Binding Protein (CIRP) in Japanese Flounder (Paralichthys olivaceus)

    Institute of Scientific and Technical Information of China (English)

    YANG Xiao; WANG Zhigang; ZHANG Quanqi; GAO Jinning; MA Liman; LI Zan; WANG Wenji; WANG Zhongkai; YU Haiyang; QI Jie; WANG Xubo

    2015-01-01

    Cold-inducible RNA-binding protein (CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first cloned from mammals. On the contrary, there are little reports in teleosts. In this study, the PoCIRP gene of the Japanese flounder was cloned and sequenced. The genomic sequence consists of seven exons and six introns. The putative PoCIRP protein of flounder was 198 amino acid residues long containing the RNA recognition motif (RRM). Phylogenetic analysis showed that the flounder PoCIRP is highly conserved with other teleost CIRPs. The 5’ flanking sequence was cloned by genome walking and many transcription factor binding sites were iden-tified. There is a CpGs region located in promoter and exon I region and the methylation state is low. Quantitative real-time PCR analysis uncovered that PoCIRP gene was widely expressed in adult tissues with the highest expression level in the ovary. The mRNA of the PoCIRP was maternally deposited and the expression level of the gene was regulated up during the gastrula and neu-rula stages. In order to gain the information how the protein interacts with mRNA, we performed the modeling of the 3D structure of the flounder PoCIRP. The results showed a cleft existing the surface of the molecular. Taken together, the results indicate that the CIRP is a multifunctional molecular in teleosts and the findings about the structure provide valuable information for understanding the basis of this protein’s function.

  7. The 25 kDa subunit of cleavage factor Im Is a RNA-binding protein that interacts with the poly(A polymerase in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Marisol Pezet-Valdez

    Full Text Available In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25 from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25 was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A polymerase (EhPAP that is responsible for the synthesis of the poly(A tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.

  8. The FTD/ALS-associated RNA-binding protein TDP-43 regulates the robustness of neuronal specification through microRNA-9a in Drosophila.

    Science.gov (United States)

    Li, Zhaodong; Lu, Yubing; Xu, Xia-Lian; Gao, Fen-Biao

    2013-01-15

    TDP-43 is an evolutionarily conserved RNA-binding protein currently under intense investigation for its involvement in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is normally localized in the nucleus, but translocated to the cytoplasm in diseased neurons. The endogenous functions of TDP-43 in the nervous system remain poorly understood. Here, we show that the loss of Drosophila TDP-43 (dTDP-43) results in an increased production of sensory bristles and sensory organ precursor (SOP) cells on the notum of some but not all flies. The location of ectopic SOPs varies among mutant flies. The penetrance of this novel phenotype is dependent on the gender and sensitive to environmental influences. A similar SOP phenotype was also observed on the wing and in the embryos. Overexpression of dTDP-43 causes both loss and ectopic production of SOPs. Ectopic expression of ALS-associated mutant human TDP-43 (hTDP-43(M337V) and hTDP-43(Q331K)) produces a less severe SOP phenotype than hTDP-43(WT), indicating a partial loss of function of mutant hTDP-43. In dTDP-43 mutants, miR-9a expression is significantly reduced. Genetic interaction studies further support the notion that dTDP-43 acts through miR-9a to control the precision of SOP specification. These findings reveal a novel role for endogenous TDP-43 in neuronal specification and suggest that the FTD/ALS-associated RNA-binding protein TDP-43 functions to ensure the robustness of genetic control programs.

  9. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

  10. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3 overexpression in pancreatic ductal adenocarcinoma correlates with poor survival

    Directory of Open Access Journals (Sweden)

    Scudamore Charles H

    2010-02-01

    Full Text Available Abstract Background Pancreatic ductal adenocarcinoma is a lethal disease with a 5-year survival rate of 4% and typically presents in an advanced stage. In this setting, prognostic markers identifying the more agrressive tumors could aid in managment decisions. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, also known as IMP3 or KOC is an oncofetal RNA-binding protein that regulates targets such as insulin-like growth factor-2 (IGF-2 and ACTB (beta-actin. Methods We evaluated the expression of IGF2BP3 by immunohistochemistry using a tissue microarray of 127 pancreatic ductal adenocarcinomas with tumor grade 1, 2 and 3 according to WHO criteria, and the prognostic value of IGF2BP3 expression. Results IGF2BP3 was found to be selectively overexpressed in pancreatic ductal adenocarcinoma tissues but not in benign pancreatic tissues. Nine (38% patient samples of tumor grade 1 (n = 24 and 27 (44% of tumor grade 2 (n = 61 showed expression of IGF2BP3. The highest rate of expression was seen in poorly differentiated specimen (grade 3, n = 42 with 26 (62% positive samples. Overall survival was found to be significantly shorter in patients with IGF2BP3 expressing tumors (P = 0.024; RR 2.3, 95% CI 1.2-4.8. Conclusions Our data suggest that IGF2BP3 overexpression identifies a subset of pancreatic ductal adenocarcinomas with an extremely poor outcome and supports the rationale for developing therapies to target the IGF pathway in this cancer.

  11. Mineralogy and thermodynamic properties of magnesium phyllosilicates formed during the alteration of a simplified nuclear glass

    Science.gov (United States)

    Debure, Mathieu; De Windt, Laurent; Frugier, Pierre; Gin, Stéphane; Vieillard, Philippe

    2016-07-01

    The precipitation of crystallized magnesium phyllosilicates generally sustains the alteration rate of nuclear waste containment glass. However, glass alteration slows down to a residual rate as soon as Mg disappears from the solution. The identification of the phyllosilicates formed is therefore crucial for modeling the long-term behavior of nuclear glass. This study deals with batch alteration of the simplified nuclear glass ISG in presence of magnesium, and the characterization of the secondary phases. Morphological, chemical and structural analyses (MET, EDX, XRD) were performed to determine the nature and structure of the precipitated phases identified as trioctahedral smectites. Analyses conducted on the secondary phases proved the presence of Al, Na and Ca in the Mg-phyllosilicate phases. Such elements had been suspected but never quantitatively measured. The experimental results were then used to determine the thermodynamic solubility constants for each precipitated secondary phase at various temperatures. The calculated values were consistent with those available for sodium and magnesium saponites in the existing thermodynamic databases.

  12. Does Addition of Propolis to Glass Ionomer Cement Alter its Physicomechanical Properties? An In Vitro Study.

    Science.gov (United States)

    Subramaniam, P; Girish Babu, K L; Neeraja, G; Pillai, S

    Propolis is a natural resinous substance produced by honey bees. The antimicrobial effects of glass ionomer cement have been shown to improve with the addition of propolis; however its effect on the physicomechanical properties of the cement is not known.

  13. Maternal mobile phone exposure alters intrinsic electrophysiological properties of CA1 pyramidal neurons in rat offspring.

    Science.gov (United States)

    Razavinasab, Moazamehosadat; Moazzami, Kasra; Shabani, Mohammad

    2016-06-01

    Some studies have shown that exposure to electromagnetic field (EMF) may result in structural damage to neurons. In this study, we have elucidated the alteration in the hippocampal function of offspring Wistar rats (n = 8 rats in each group) that were chronically exposed to mobile phones during their gestational period by applying behavioral, histological, and electrophysiological tests. Rats in the EMF group were exposed to 900 MHz pulsed-EMF irradiation for 6 h/day. Whole cell recordings in hippocampal pyramidal cells in the mobile phone groups did show a decrease in neuronal excitability. Mobile phone exposure was mostly associated with a decrease in the number of action potentials fired in spontaneous activity and in response to current injection in both male and female groups. There was an increase in the amplitude of the afterhyperpolarization (AHP) in mobile phone rats compared with the control. The results of the passive avoidance and Morris water maze assessment of learning and memory performance showed that phone exposure significantly altered learning acquisition and memory retention in male and female rats compared with the control rats. Light microscopy study of brain sections of the control and mobile phone-exposed rats showed normal morphology.Our results suggest that exposure to mobile phones adversely affects the cognitive performance of both female and male offspring rats using behavioral and electrophysiological techniques.

  14. Biochemical evaluation of a 108-member deglycobleomycin library: viability of a selection strategy for identifying bleomycin analogues with altered properties.

    Science.gov (United States)

    Ma, Qian; Xu, Zhidong; Schroeder, Benjamin R; Sun, Wenyue; Wei, Fang; Hashimoto, Shigeki; Konishi, Kazuhide; Leitheiser, Christopher J; Hecht, Sidney M

    2007-10-17

    The bleomycins (BLMs) are clinically used glycopeptide antitumor antibiotics that have been shown to mediate the sequence-selective oxidative damage of both DNA and RNA. Previously, we described the solid-phase synthesis of a library of 108 unique analogues of deglycoBLM A6, a congener that cleaves DNA analogously to BLM itself. Each member of the library was assayed for its ability to effect single- and double-strand nicking of duplex DNA, sequence-selective DNA cleavage, and RNA cleavage in the presence and absence of a metal ion cofactor. All of the analogues tested were found to mediate concentration-dependent plasmid DNA relaxation to some extent, and a number exhibited double-strand cleavage with an efficiency comparable to or greater than deglycoBLM A6. Further, some analogues having altered linker and metal-binding domains mediated altered sequence-selective cleavage, and a few were found to cleave a tRNA3Lys transcript both in the presence and in the absence of a metal cofactor. The results provide insights into structural elements within BLM that control DNA and RNA cleavage. The present study also permits inferences to be drawn regarding the practicality of a selection strategy for the solid-phase construction and evaluation of large libraries of BLM analogues having altered properties.

  15. Alteration of corrosion and nanomechanical properties of pulse electrodeposited Ni/SiC nanocomposite coatings

    Energy Technology Data Exchange (ETDEWEB)

    Zarghami, V. [Department of Materials Science and Engineering, Sharif University of Technology, Azadi Street, Tehran (Iran, Islamic Republic of); Ghorbani, M., E-mail: Ghorbani@sharif.edu [Department of Materials Science and Engineering, Sharif University of Technology, Azadi Street, Tehran (Iran, Islamic Republic of); Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Azadi Street, Tehran (Iran, Islamic Republic of)

    2014-06-15

    Highlights: • Preparing Ni/SiC coatings on the Cu substrate by using of rotating disk electrode. • Optimizing of pulse current density parameters. • Optimizing of SiC content in the bath. • Investigation the effect of codeposited SiC amount on the properties of coatings. - Abstract: Nickel/silicon carbide composite electrodeposits were prepared on a rotating disk electrode (RDE), under pulse current condition. The effect of pulse parameters, current density, SiC content in the electrolyte on the codeposition of SiC were studied. Afterwards, the effect of codeposited SiC amount was investigated on electrochemical behavior and nanomechanical properties of coatings. The coatings were analyzed with Scanning Electron Microscopy (SEM), linear polarization, nanoindentation and Atomic Force Microscopy (AFM). The Ni–SiC electrocomposites, prepared at optimum conditions, exhibited improved nanomechanical properties in comparison to pure nickel electrodeposits. With increasing current density the morphology changed from flat surface to cauliflower structure. The Ni–SiC electrocomposites exhibited improved nanomechanical properties and corrosion resistances in comparison to pure nickel electrodeposits and these properties were improving with increasing codeposited SiC particles in electrocomposites.

  16. Dynamic Alterations in Microarchitecture, Mineralization and Mechanical Property of Subchondral Bone in Rat Medial Meniscal Tear Model of Osteoarthritis

    Directory of Open Access Journals (Sweden)

    De-Gang Yu

    2015-01-01

    Full Text Available Background: The properties of subchondral bone influence the integrity of articular cartilage in the pathogenesis of osteoarthritis (OA. However, the characteristics of subchondral bone alterations remain unresolved. The present study aimed to observe the dynamic alterations in the microarchitecture, mineralization, and mechanical properties of subchondral bone during the progression of OA. Methods: A medial meniscal tear (MMT operation was performed in 128 adult Sprague Dawley rats to induce OA. At 2, 4, 8, and 12 weeks following the MMT operation, cartilage degeneration was evaluated using toluidine blue O staining, whereas changes in the microarchitecture indices and tissue mineral density (TMD, mineral-to-collagen ratio, and intrinsic mechanical properties of subchondral bone plates (BPs and trabecular bones (Tbs were measured using micro-computed tomography scanning, confocal Raman microspectroscopy and nanoindentation testing, respectively. Results: Cartilage degeneration occurred and worsened progressively from 2 to 12 weeks after OA induction. Microarchitecture analysis revealed that the subchondral bone shifted from bone resorption early (reduced trabecular BV/TV, trabecular number, connectivity density and trabecular thickness [Tb.Th], and increased trabecular spacing (Tb.Sp at 2 and 4 weeks to bone accretion late (increased BV/TV, Tb.Th and thickness of subchondral bone plate, and reduced Tb.Sp at 8 and 12 weeks. The TMD of both the BP and Tb displayed no significant changes at 2 and 4 weeks but decreased at 8 and 12 weeks. The mineral-to-collagen ratio showed a significant decrease from 4 weeks for the Tb and from 8 weeks for the BP after OA induction. Both the elastic modulus and hardness of the Tb showed a significant decrease from 4 weeks after OA induction. The BP showed a significant decrease in its elastic modulus from 8 weeks and its hardness from 4 weeks. Conclusion: The microarchitecture, mineralization and mechanical

  17. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A;

    1988-01-01

    In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...

  18. Fatigue Induced Alteration of the Superficial Strength Properties of 2024 Aluminum Alloy

    Institute of Scientific and Technical Information of China (English)

    K.-D. Bouzakis; I. Mirisidis; Sp. G. Pantelakis; A.N. Chamos

    2011-01-01

    aluminum alloy 2024 T3 specimens have been subjected to constant amplitude fatigue loading at R=0.1. During fatigue, an appreciable increase of the surface hardness of the material at the meso-scale can be observed and captured by means of nanoindentations. Surface hardness increases with increasing fatigue stress amplitude and advancing number of applied fatigue cycles. Observed increase of specimen surface hardening degree during fatigue causes an evolution of superficial mechanical strength properties of the alloy. Stress-strain curves associated with the evoluting superficial mechanical properties are derived, employing a developed finite element method (FEM)-supported evaluation procedure of nanoindentation experimental results.

  19. Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization.

    Directory of Open Access Journals (Sweden)

    Arthur S Kim

    2014-07-01

    Full Text Available Human antibody 10E8 targets the conserved membrane proximal external region (MPER of envelope glycoprotein (Env subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.

  20. Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata

    Science.gov (United States)

    Shaik, Noor F.; Neal, Erin M.; Leone, Sarah G.; Cali, Brian J.; Peel, Michael T.; Grannas, Amanda M.; Wykoff, Dennis D.

    2016-01-01

    The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine. PMID:27015653

  1. Cytomechanical properties of papaver pollen tubes are altered after self-incompatibility challenge

    NARCIS (Netherlands)

    Geitmann, A.; McConnaughey, W.; Lang-Pauluzzi, I.; Franklin-Tong, V.E.; Emons, A.M.C.

    2004-01-01

    Self-incompatibility (SI) in Papaver rhoeas triggers a ligand-mediated signal transduction cascade, resulting in the inhibition of incompatible pollen tube growth. Using a cytomechanical approach we have demonstrated that dramatic changes to the mechanical properties of incompatible pollen tubes are

  2. Induction of advanced glycation end products and alterations of the tensile properties of articular cartilage

    NARCIS (Netherlands)

    Chen, A.C.; Temple, M.M.; Ng, D.M.; Verzijl, N.; Groot, J. de; TeKoppele, J.M.; Sah, R.L.

    2002-01-01

    Objective. To determine whether increasing advanced glycation end products (AGEs) in bovine articular cartilage to levels present in aged human cartilage modulates the tensile biomechanical properties of the tissue. Methods. Adult bovine articular cartilage samples were incubated in a buffer solutio

  3. Differences in Age-Related Alterations in Muscle Contraction Properties in Rat Tongue and Hindlimb

    Science.gov (United States)

    Connor, Nadine P.; Ota, Fumikazu; Nagai, Hiromi; Russell, John A.; Leverson, Glen

    2008-01-01

    Purpose: Because of differences in muscle architecture and biomechanics, the purpose of this study was to determine whether muscle contractile properties of rat hindlimb and tongue were differentially affected by aging. Method: Deep peroneal and hypoglossal nerves were stimulated in 6 young and 7 old Fischer 344-Brown Norway rats to allow…

  4. Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3.

    Directory of Open Access Journals (Sweden)

    Julie Pilotte

    Full Text Available MicroRNAs (miRNAs play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ~70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ~70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of

  5. Genetic polymorphisms in the microRNA binding-sites of the thymidylate synthase gene predict risk and survival in gastric cancer.

    Science.gov (United States)

    Shen, Rong; Liu, Hongliang; Wen, Juyi; Liu, Zhensheng; Wang, Li-E; Wang, Qiming; Tan, Dongfeng; Ajani, Jaffer A; Wei, Qingyi

    2015-09-01

    Thymidylate synthase (TYMS) plays a crucial role in folate metabolism as well as DNA synthesis and repair. We hypothesized that functional polymorphisms in the 3' UTR of TYMS are associated with gastric cancer risk and survival. In the present study, we tested our hypothesis by genotyping three potentially functional (at miRNA binding sites) TYMS SNPs (rs16430 6bp del/ins, rs2790 A>G and rs1059394 C>T) in 379 gastric cancer patients and 431 cancer-free controls. Compared with the rs16430 6bp/6bp + 6bp/0bp genotypes, the 0bp/0bp genotype was associated with significantly increased gastric cancer risk (adjusted OR = 1.72, 95% CI = 1.15-2.58). Similarly, rs2790 GG and rs1059394 TT genotypes were also associated with significantly increased risk (adjusted OR = 2.52, 95% CI = 1.25-5.10 and adjusted OR = 1.57, 95% CI = 1.04-2.35, respectively), compared with AA + AG and CC + CT genotypes, respectively. In the haplotype analysis, the T-G-0bp haplotype was associated with significantly increased gastric cancer risk, compared with the C-A-6bp haplotype (adjusted OR = 1.34, 95% CI = 1.05-1.72). Survival analysis revealed that rs16430 0bp/0bp and rs1059394 TT genotypes were also associated with poor survival in gastric cancer patients who received chemotherapy treatment (adjusted HR = 1.61, 95% CI = 1.05-2.48 and adjusted HR = 1.59, 95% CI = 1.02-2.48, respectively). These results suggest that these three variants in the miRNA binding sites of TYMS may be associated with cancer risk and survival of gastric cancer patients. Larger population studies are warranted to verify these findings.

  6. The RNA-binding Protein TDP-43 Selectively Disrupts MicroRNA-1/206 Incorporation into the RNA-induced Silencing Complex*♦

    Science.gov (United States)

    King, Isabelle N.; Yartseva, Valeria; Salas, Donaldo; Kumar, Abhishek; Heidersbach, Amy; Ando, D. Michael; Stallings, Nancy R.; Elliott, Jeffrey L.; Srivastava, Deepak; Ivey, Kathryn N.

    2014-01-01

    MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, we screened for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. Here, we describe a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced dmiR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. Our findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity. PMID:24719334

  7. Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

    Science.gov (United States)

    Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2013-12-01

    The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.

  8. CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Hidehito Kuroyanagi

    Full Text Available An enormous number of alternative pre-mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a-4c and 7a-7b, of the Caenorhabditis elegans uncoordinated (unc-32 gene, encoding the a subunit of V0 complex of vacuolar-type H(+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA-binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA-binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive

  9. Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral

    Science.gov (United States)

    Paschalis, E.P.; Tatakis, D.N.; Robins, S.; Fratzl, P.; Manjubala, I.; Zoehrer, R.; Gamsjaeger, S.; Buchinger, B.; Roschger, A.; Phipps, R.; Boskey, A.L.; Dall'Ara, E.; Varga, P.; Zysset, P.; Klaushofer, K.; Roschger, P.

    2011-01-01

    In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and β-APN treated animals were fed additionally with β-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2–L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that β-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p < 0.05). Further, compression tests revealed a significant negative impact of β-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, β-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence

  10. Induction of advanced glycation end products and alterations of the tensile properties of articular cartilage

    OpenAIRE

    Chen, A C; Temple, M.M.; Ng, D.M.; Verzijl, N; de Groot, J.; TeKoppele, J.M.; Sah, R.L.

    2002-01-01

    Objective. To determine whether increasing advanced glycation end products (AGEs) in bovine articular cartilage to levels present in aged human cartilage modulates the tensile biomechanical properties of the tissue. Methods. Adult bovine articular cartilage samples were incubated in a buffer solution with ribose to induce the formation of AGEs or in a control solution. Portions of cartilage samples were assayed for biochemical indices of AGEs and tested to assess their tensile biomechanical p...

  11. RNA editing of the GABAA receptor α3 subunit alters the functional properties of recombinant receptors

    OpenAIRE

    Nimmich, Mitchell L.; Heidelberg, Laura S.; Fisher, Janet L.

    2009-01-01

    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors co...

  12. Computer analysis of effects of altering jet fuel properties on refinery costs and yields

    Science.gov (United States)

    Breton, T.; Dunbar, D.

    1984-01-01

    This study was undertaken to evaluate the adequacy of future U.S. jet fuel supplies, the potential for large increases in the cost of jet fuel, and to what extent a relaxation in jet fuel properties would remedy these potential problems. The results of the study indicate that refiners should be able to meet jet fuel output requirements in all regions of the country within the current Jet A specifications during the 1990-2010 period. The results also indicate that it will be more difficult to meet Jet A specifications on the West Coast, because the feedstock quality is worse and the required jet fuel yield (jet fuel/crude refined) is higher than in the East. The results show that jet fuel production costs could be reduced by relaxing fuel properties. Potential cost savings in the East (PADDs I-IV) through property relaxation were found to be about 1.3 cents/liter (5 cents/gallon) in January 1, 1981 dollars between 1990 and 2010. However, the savings from property relaxation were all obtained within the range of current Jet A specifications, so there is no financial incentive to relax Jet A fuel specifications in the East. In the West (PADD V) the potential cost savings from lowering fuel quality were considerably greater than in the East. Cost savings from 2.7 to 3.7 cents/liter (10-14 cents/gallon) were found. In contrast to the East, on the West Coast a significant part of the savings was obtained through relaxation of the current Jet A fuel specifications.

  13. Polymeric tannins significantly alter properties and in vitro digestibility of partially gelatinized intact starch granule.

    Science.gov (United States)

    Amoako, Derrick B; Awika, Joseph M

    2016-10-01

    Excess calorie intake is a growing global problem. This study investigated effect of complexing partially gelatinized starch with condensed tannins on in vitro starch digestibility. Extracts from tannin and non-tannin sorghum, and cellulose control, were reacted with normal and waxy maize starch in 30% (30E) and 50% ethanol (50E) solutions at 70°C/20min. More tannins complexed with the 30E than 50E starches (mean 6.2 vs 3.5mg/g, respectively). In the 30E treatments, tannins significantly increased crystallinity, pasting temperature, peak viscosity, and slow digesting starch (from 100 to 274mg/g) in normal, but not waxy starch, suggesting intragranular cross-linking with amylose. Tannins doubled resistant starch (RS) to approx. 300mg/g in both starches. In 50E treatments, tannins made both maize starches behave like raw potato starch (>90% RS), suggesting granule surface interactions dominated. Non-tannin treatments generally behaved similar to cellulose. Condensed tannins could be used to favorably alter starch digestion profile.

  14. Altered small-world properties of gray matter networks in breast cancer

    Directory of Open Access Journals (Sweden)

    Hosseini S M

    2012-05-01

    Full Text Available Abstract Background Breast cancer survivors, particularly those treated with chemotherapy, are at significantly increased risk for long-term cognitive and neurobiologic impairments. These deficits tend to involve skills that are subserved by distributed brain networks. Additionally, neuroimaging studies have shown a diffuse pattern of brain structure changes in chemotherapy-treated breast cancer survivors that might impact large-scale brain networks. Methods We therefore applied graph theoretical analysis to compare the gray matter structural networks of female breast cancer survivors with a history of chemotherapy treatment and healthy age and education matched female controls. Results Results revealed reduced clustering coefficient and small-world index in the brain network of the breast cancer patients across a range of network densities. In addition, the network of the breast cancer group had less highly interactive nodes and reduced degree/centrality in the frontotemporal regions compared to controls, which may help explain the common impairments of memory and executive functioning among these patients. Conclusions These results suggest that breast cancer and chemotherapy may decrease regional connectivity as well as global network organization and integration, reducing efficiency of the network. To our knowledge, this is the first report of altered large-scale brain networks associated with breast cancer and chemotherapy.

  15. Functional and structural alterations of epithelial barrier properties of rat ileum following X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Dublineau, I. [Inst. de Radioprotection et de Surete Nucleaire (IRSN), Direction de la RadioProtection de l' Homme, Service de Radiobiologie et d' Epidemiologie, Fontenay-aux-Roses, CEDEX (France)]. E-mail: isabelle.dublineau@irsn.fr; Lebrun, F. [Commissariat a l' Energie Atomique (CEA), Dept. de Radiopathologie et de Radiobiologie, Fontenay-aux-Roses, CEDEX (France); Grison, S.; Griffiths, N.M. [Inst. de Radioprotection et de Surete Nucleaire (IRSN), Direction de la RadioProtection de l' Homme, Service de Radiobiologie et d' Epidemiologie, Fontenay-aux-Roses, CEDEX (France)

    2004-02-01

    Irradiation of the digestive system leads to alterations of the small intestine. We have characterized the disruption of the barrier integrity in rat ileum from 1 to 14 days following irradiation ranging from 6 to 12 Gy. The intestinal permeability to {sup 14}C-mannitol and {sup 3}H-dextran 70,000 was measured in vitro in Ussing chambers. In parallel to these functional studies, immunohistochemical analyses of junctional proteins (ZO-1 and {beta}-catenin) of ileal epithelium were performed by confocal microscopy. Irradiation with 10 Gy induced a marked decrease in epithelial tissue resistance at three days and a fivefold increase in mannitol permeability, without modifications of dextran permeability. A disorganization of the localization for ZO-1 and {beta}-catenin was also observed. At 7 days after irradiation, we observed a recovery of the organization of junctional proteins in parallel to a return of intestinal permeability to control value. In addition to these time-dependent effects, a gradual effect on epithelial integrity of the radiation doses was observed 3 days after irradiation. This study shows a disruption of the integrity of the intestinal barrier in rat ileum following abdominal X-irradiation, depending on the time postirradiation and on the delivered dose. The loss of barrier integrity was characterized by a disorganization of proteins of tight and adherent junctions, leading to increased intestinal permeability to mannitol. (author)

  16. CERKL, a retinal disease gene, encodes an mRNA-binding protein that localizes in compact and untranslated mRNPs associated with microtubules.

    Directory of Open Access Journals (Sweden)

    Alihamze Fathinajafabadi

    Full Text Available The function of CERKL (CERamide Kinase Like, a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.

  17. Insulin-like growth factor II mRNA binding protein 3 (IMP3 is overexpressed in prostate cancer and correlates with higher Gleason scores

    Directory of Open Access Journals (Sweden)

    Mortezavi Ashkan

    2010-06-01

    Full Text Available Abstract Background The oncofetal protein insulin-like growth factor II mRNA binding protein 3 (IMP3 is an important factor for cell-migration and adhesion in malignancies. Recent studies have shown a remarkable overexpression of IMP3 in different human malignant neoplasms and also revealed it as an important prognostic marker in some tumor entities. To our knowledge, IMP3 expression has not been investigated in prostate carcinomas so far. Methods Immunohistochemical stainings for IMP3 were performed on tissue microarray (TMA organized samples from 507 patients: 31 normal prostate tissues, 425 primary carcinomas and 51 prostate cancer metastases or castration-resistant prostate cancers (CRPC. IMP3 immunoreactivity was semiquantitatively scored and correlated with clinical-pathologic parameters including survival. Results IMP3 is significantly stronger expressed in prostate carcinomas compared to normal prostate tissues (p Conclusions Although IMP3 is overexpressed in a significant proportion of prostate cancer cases, which might be of importance for novel therapeutic approaches, it does not appear to possess any immediate diagnostic or prognostic value, limiting its potential as a tissue biomarker for prostate cancer. These results might be corroborated by the fact, that two independent tumor cohorts were separately reviewed.

  18. Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Hamada, Junichi; Shoda, Katsutoshi; Masuda, Kiyoshi; Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

    2016-03-29

    T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC.

  19. C. elegans RNA-binding protein GLD-1 recognizes its multiple targets using sequence, context, and structural information to repress translation.

    Science.gov (United States)

    Doh, Jung H; Jung, Yuchae; Reinke, Valerie; Lee, Min-Ho

    2013-10-01

    Caenorhabditis elegans GLD-1, a maxi-KH motif containing RNA-binding protein, has various functions mainly during female germ cell development, suggesting that it likely controls the expression of a selective group of maternal mRNAs. To gain an insight into how GLD-1 specifically recognizes these mRNA targets, we identified 38 biochemically proven GLD-1 binding regions from multiple mRNA targets that are among over 100 putative targets co-immunoprecipitated with GLD-1. The sequence information of these regions revealed three over-represented and phylogenetically conserved sequence motifs. We found that two of the motifs, one of which is novel, are important for GLD-1 binding in several GLD-1 binding regions but not in other regions. Further analyses indicate that the importance of one of the sequence motifs is dependent on two aspects: (1) surrounding sequence information, likely acting as an accessory feature for GLD-1 to efficiently select the sequence motif and (2) RNA secondary structural environment where the sequence motif resides, which likely provides "binding-site accessibility" for GLD-1 to effectively recognize its targets. Our data suggest some mRNAs recruit GLD-1 by a distinct mechanism, which involves more than one sequence motif that needs to be embedded in the correct context and structural environment.

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Nucleolar accumulation of RNA binding proteins induced by Actinomycin D is functional in Trypanosoma cruzi and Leishmania mexicana but not in T. brucei.

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    Ezequiel Názer

    Full Text Available We have recently shown in T. cruzi that a group of RNA Binding Proteins (RBPs, involved in mRNA metabolism, are accumulated into the nucleolus in response to Actinomycin D (ActD treatment. In this work, we have extended our analysis to other members of the trypanosomatid lineage. In agreement with our previous study, the mechanism seems to be conserved in L. mexicana, since both endogenous RBPs and a transgenic RBP were relocalized to the nucleolus in parasites exposed to ActD. In contrast, in T. brucei, neither endogenous RBPs (TbRRM1 and TbPABP2 nor a transgenic RBP from T. cruzi were accumulated into the nucleolus under such treatment. Interestingly, when a transgenic TbRRM1 was expressed in T. cruzi and the parasites exposed to ActD, TbRRM1 relocated to the nucleolus, suggesting that it contains the necessary sequence elements to be targeted to the nucleolus. Together, both experiments demonstrate that the mechanism behind nucleolar localization of RBPs, which is present in T. cruzi and L. mexicana, is not functional in T. brucei, suggesting that it has been lost or retained differentially during the evolution of the trypanosomatid lineage.

  2. Identification and Evaluation of Novel Drug Targets against the Human Fungal Pathogen Aspergillus fumigatus with Elaboration on the Possible Role of RNA-Binding Protein

    Science.gov (United States)

    Malekzadeh, Saeid; Sardari, Soroush; Azerang, Parisa; Khorasanizadeh, Dorsa; Amiri, Solmaz Agha; Azizi, Mohammad; Mohajerani, Nazanin; Khalaj, Vahid

    2017-01-01

    Bakground: Aspergillus fumigatus is an airborne opportunistic fungal pathogen that can cause fatal infections in immunocompromised patients. Although the current anti-fungal therapies are relatively efficient, some issues such as drug toxicity, drug interactions, and the emergence of drug-resistant fungi have promoted the intense research toward finding the novel drug targets. Methods: In search of new antifungal drug targets, we have used a bioinformatics approach to identify novel drug targets. We compared the whole proteome of this organism with yeast Saccharomyces cerevisiae to come up with 153 specific proteins. Further screening of these proteins revealed 50 potential molecular targets in A. fumigatus. Amongst them, RNA-binding protein (RBP) was selected for further examination. The aspergillus fumigatus RBP (AfuRBP), as a peptidylprolyl isomerase, was evaluated by homology modeling and bioinformatics tools. RBP-deficient mutant strains of A. fumigatus were generated and characterized. Furthermore, the susceptibility of these strains to known peptidylprolyl isomerase inhibitors was assessed. Results: AfuRBP-deficient mutants demonstrated a normal growth phenotype. MIC assay results using inhibitors of peptidylprolyl isomerase confirmed a higher sensitivity of these mutants compared to the wild type. Conclusion: Our bioinformatics approach revealed a number of fungal-specific proteins that may be considered as new targets for drug discovery purposes. Peptidylprolyl isomerase, as a possible drug target, was evaluated against two potential inhibitors, and the promising results were investigated mechanistically. Future studies would confirm the impact of such target on the antifungal discovery investigations PMID:28000798

  3. A Puf RNA-binding protein encoding gene PlM90 regulates the sexual and asexual life stages of the litchi downy blight pathogen Peronophythora litchii.

    Science.gov (United States)

    Jiang, Liqun; Ye, Wenwu; Situ, Junjian; Chen, Yubin; Yang, Xinyu; Kong, Guanghui; Liu, Yaya; Tinashe, Runyanga J; Xi, Pinggen; Wang, Yuanchao; Jiang, Zide

    2017-01-01

    Sexual and asexual reproduction are two key processes in the pathogenic cycle of many filamentous pathogens. However in Peronophythora litchii, the causal pathogen for the litchi downy blight disease, critical regulator(s) of sexual or asexual differentiation has not been elucidated. In this study, we cloned a gene named PlM90 from P. litchii, which encodes a putative Puf RNA-binding protein. We found that PlM90 was highly expressed during asexual development, and much higher than that during sexual development, while relatively lower during cyst germination and plant infection. By polyethylene glycol (PEG)-mediated protoplast transformation, we generated three PlM90-silenced transformants and found a severely impaired ability in sexual spore production and a delay in stages of zoospore release and encystment. However, the pathogenicity of P. litchii was not affected by PlM90-silencing. Therefore we conclude that PlM90 specifically regulates the sexual and asexual differentiation of P. litchii.

  4. In various protein complexes, disordered protomers have large per-residue surface areas and area of protein-, DNA- and RNA-binding interfaces.

    Science.gov (United States)

    Wu, Zhonghua; Hu, Gang; Yang, Jianyi; Peng, Zhenling; Uversky, Vladimir N; Kurgan, Lukasz

    2015-09-14

    We provide first large scale analysis of the peculiarities of surface areas of 5658 dissimilar (below 50% sequence similarity) proteins with known 3D-structures that bind to proteins, DNA or RNAs. We show here that area of the protein surface is highly correlated with the protein length. The size of the interface surface is only modestly correlated with the protein size, except for RNA-binding proteins where larger proteins are characterized by larger interfaces. Disordered proteins with disordered interfaces are characterized by significantly larger per-residue areas of their surfaces and interfaces when compared to the structured proteins. These result are applicable for proteins involved in interaction with DNA, RNA, and proteins and suggest that disordered proteins and binding regions are less compact and more likely to assume extended shape. We demonstrate that disordered protein binding residues in the interfaces of disordered proteins drive the increase in the per residue area of these interfaces. Our results can be used to predict in silico whether a given protomer from the DNA, RNA or protein complex is likely to be disordered in its unbound form.

  5. Acute reduction of neuronal RNA binding Elavl2 protein and Gap43 mRNA in mouse hippocampus after kainic acid treatment.

    Science.gov (United States)

    Ohtsuka, Takafumi; Yano, Masato; Okano, Hideyuki

    2015-10-09

    Activity-dependent gene regulation in neurons has been hypothesized to be under transcriptional control and to include dramatic increases in immediate early genes (IEGs) after neuronal activity. In addition, several reports have focused on post-transcriptional regulation, which could be mediated by neuronal post-transcriptional regulators, including RNA binding proteins (RNABPs). One such protein family is the neuronal Elavls (nElavls; Elavl2, Elavl3, and Elavl4), whose members are widely expressed in peripheral and central nervous system. Previous reports showed that Elavl3 and 4 are up-regulated following repeated stimulation such as during cocaine administration, a seizure, or a spatial discrimination task. In this study, we focused on Elavl2, a candidate gene for schizophrenia and studied its role in neuronal activity. First we found that Elavl2 has a cell-type specific expression pattern that is highly expressed in hippocampal CA3 pyramidal neurons and hilar interneurons using Elavl2 specific antibody. Second, unexpectedly, we discovered that the Elavl2 protein level in the hippocampus was acutely down-regulated for 3 h after a kainic acid (KA)-induced seizure in the hippocampal CA3 region. In addition, level of Gap43 mRNA, a target mRNA of Elavl2 is decreased 12 h after KA treatment, thus suggesting the involvement of Elavl2 in activity-dependent RNA regulation.

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

    Directory of Open Access Journals (Sweden)

    Deivid C. Rodrigues

    2016-10-01

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

  7. Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

    Science.gov (United States)

    Edinger, Nufar; Lebendiker, Mario; Klein, Shoshana; Zigler, Maya; Langut, Yael; Levitzki, Alexander

    2016-01-01

    Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC. PMID:27598772

  8. Cold-Inducible RNA-Binding Protein Bypasses Replicative Senescence in Primary Cells through Extracellular Signal-Regulated Kinase 1 and 2 Activation▿ †

    Science.gov (United States)

    Artero-Castro, Ana; Callejas, Francisco B.; Castellvi, Josep; Kondoh, Hiroshi; Carnero, Amancio; Fernández-Marcos, Pablo J.; Serrano, Manuel; Ramón y Cajal, Santiago; Lleonart, Matilde E.

    2009-01-01

    Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16INK4a, p21WAF1, and p19ARF upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research. PMID:19158277

  9. The effects of Hedgehog on the RNA-binding protein Msi1 in the proliferation and apoptosis of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    In-Sun Hong

    Full Text Available Human umbilical cord blood (UCB-derived mesenchymal stem cells (MSCs are essential tools for regenerative medicine due to their capacity for self-renewal and multi-lineage differentiation. As MSCs are found in very small numbers in various tissues, in vitro cell expansion is an essential step that is needed before these cells can be used in clinical applications. Therefore, it is important to identify and characterize factors that are involved in MSC proliferation and apoptosis. In the present study, we focused on Hedgehog (Hh signaling because several studies have proposed that Hh signaling plays a critical role in controlling the proliferation of stem and progenitor cells. However, the molecular mechanisms underlying the effects on the proliferation and apoptosis of MSCs remain unclear. In this study, we evaluated the direct effects of Hh signaling on the proliferation and apoptosis of hUCB-MSCs as well as investigated potential downstream regulatory mechanisms that may be responsible for Hh signaling. We observed that the Hedgehog agonist purmorphamine enhanced cell proliferation and suppressed apoptosis through the RNA-binding protein Msi1 by regulating the expression of an oncoprotein (i.e., c-Myc, a cell cycle regulatory molecule (i.e., p21(CIP1,WAF1 and two microRNAs (i.e., miRNA-148a and miRNA-148b. This study provides novel insights into the molecular mechanisms regulating the self-renewal capability of MSCs with relevance to clinical applications.

  10. Rbms3, an RNA-binding protein, mediates the expression of Ptf1a by binding to its 3'UTR during mouse pancreas development.

    Science.gov (United States)

    Lu, Chung-Kuang; Lai, Yi-Chyi; Chen, Hau-Ren; Chiang, Ming-Ko

    2012-07-01

    The development of the pancreas is a complicated process that is regulated on several levels. Pancreas transcription factor 1, alpha subunit (Ptf1a), also known as p48, is a pancreas-specific basic helix-loop-helix transcription factor that is critical for both exocrine pancreas development and maintenance of acinar cell differentiation. Based on a differential screening assay, we identified Rbms3, a gene encoding a glycine-rich RNA-binding protein, to be specifically expressed in the neural tube and the pancreatic rudiment of e10.5 embryos. The presence of Rbms3 in the early developing pancreas suggests that specific post-transcriptional regulation mechanisms play an important role in controlling pancreas development. In this study, we show that Rbms3 binds to the 3'UTR of Ptf1a mRNA, but not the 3'UTR of Pdx1, which is another pancreatic transcription factor. The ectopic expression of Rbms3 stimulates the translation of a reporter gene carrying the Ptf1a 3'UTR. In addition, when Rbms3 expression is suppressed in the AR42J-B13 pancreatic exocrine cell line, the expression of Ptf1a is also down-regulated. These results suggest that binding of Rbms3 to the 3'UTR of Ptf1a regulates the production of the Ptf1a protein and, thereby, indirectly regulates the expression of the Ptf1a downstream target genes.

  11. The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and β-catenin protein expression.

    Science.gov (United States)

    de Bruin, Ruben G; van der Veer, Eric P; Prins, Jurriën; Lee, Dae Hyun; Dane, Martijn J C; Zhang, Huayu; Roeten, Marko K; Bijkerk, Roel; de Boer, Hetty C; Rabelink, Ton J; van Zonneveld, Anton Jan; van Gils, Janine M

    2016-02-24

    Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and β-catenin and can induce mRNA translation mediated by the 3'UTR of these genes. Reduced quaking levels attenuated VE-cadherin and β-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity.

  12. Adding energy minimization strategy to peptide-design algorithm enables better search for RNA-binding peptides: Redesigned λ N peptide binds boxB RNA.

    Science.gov (United States)

    Xiao, Xingqing; Hung, Michelle E; Leonard, Joshua N; Hall, Carol K

    2016-10-15

    Our previously developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc.

  13. Effect of proline rich domain of an RNA-binding protein Sam68 in cell growth process, death and B cell signal transduction

    Institute of Scientific and Technical Information of China (English)

    LI Qing-hua; FAN Tian-xue; PANG Tian-xiang; YUAN Wen-su; HAN Zhong-chao

    2006-01-01

    Background Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line.Methods By using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometfic analyses were performed to investigate the Sam68 functions.Results A slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells.Conclusions The proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

  14. Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Zhe Geng

    2015-01-01

    Full Text Available RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1.

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

    Science.gov (United States)

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

    2017-01-01

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

  16. Doping of TiO2 Polymorphs for Altered Optical and Photocatalytic Properties

    Directory of Open Access Journals (Sweden)

    Xiliang Nie

    2009-01-01

    Full Text Available This paper reviews recent investigations of the influence of dopants on the optical properties of TiO2 polymorphs. The common undoped polymorphs of TiO2 are discussed and compared. The results of recent doping efforts are tabulated, and discussed in the context of doping by elements of the same chemical group. Dopant effects on the band gap and photocatalytic activity are interpreted with reference to a simple qualitative picture of the TiO2 electronic structure, which is supported with first-principles calculations.

  17. Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs.

    Science.gov (United States)

    Boyen, F; Haesebrouck, F; Vanparys, A; Volf, J; Mahu, M; Van Immerseel, F; Rychlik, I; Dewulf, J; Ducatelle, R; Pasmans, F

    2008-12-10

    Salmonella Typhimurium infections in pigs are a major source of human foodborne salmonellosis. To reduce the number of infected pigs, acidification of feed or drinking water is a common practice. The aim of the present study was to determine whether some frequently used short- (SCFA) and medium-chain fatty acids (MCFA) are able to alter virulence gene expression and to decrease Salmonella Typhimurium colonization and shedding in pigs using well established and controlled in vitro and in vivo assays. Minimal inhibitory concentrations (MIC) of 4 SCFA (formic acid, acetic acid, propionic acid and butyric acid) and 2 MCFA (caproic and caprylic acid) were determined using 54 porcine Salmonella Typhimurium field strains. MIC values increased at increasing pH-values and were two to eight times lower for MCFA than for SCFA. Expression of virulence gene fimA was significantly lower when bacteria were grown in LB-broth supplemented with sub-MIC concentrations of caproic or caprylic acid (2 mM). Expression of hilA and invasion in porcine intestinal epithelial cells was significantly lower when bacteria were grown in LB-broth containing sub-MIC concentrations of butyric acid or propionic acid (10 mM) and caproic or caprylic acid (2 mM). When given as feed supplement to pigs experimentally infected with Salmonella Typhimurium, coated butyric acid decreased the levels of faecal shedding and intestinal colonization, but had no influence on the colonization of tonsils, spleen and liver. Uncoated fatty acids, however, did not influence fecal shedding, intestinal or tonsillar colonization in pigs. In conclusion, supplementing feed with certain coated fatty acids, such as butyric acid, may help to reduce the Salmonella load in pigs.

  18. Alterations in the properties of neonatal thalamocortical synapses with time in in vitro slices.

    Science.gov (United States)

    Luz, Liliana L; Currie, Stephen P; Daw, Michael I

    2017-01-01

    New synapses are constantly being generated and lost in the living brain with only a subset of these being stabilized to form an enduring component of neuronal circuitry. The properties of synaptic transmission have primarily been established in a variety of in vitro neuronal preparations. It is not clear, however, if newly-formed and persistent synapses contribute to the results of these studies consistently throughout the lifespan of these preparations. In neonatal somatosensory, barrel, cortex we have previously hypothesized that a population of thalamocortical synapses displaying unusually slow kinetics represent newly-formed, default-transient synapses. This clear phenotype would provide an ideal tool to investigate if such newly formed synapses consistently contribute to synaptic transmission throughout a normal experimental protocol. We show that the proportion of synapses recorded in vitro displaying slow kinetics decreases with time after brain slice preparation. However, slow synapses persist in vitro in the presence of either minocycline, an inhibitor of microglia-mediated synapse elimination, or the TrkB agonist 7,8-dihydroxyflavone a promoter of synapse formation. These findings show that the observed properties of synaptic transmission may systematically change with time in vitro in a standard brain slice preparation.

  19. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    Science.gov (United States)

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

  20. Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Alterations of membrane lipid biophysical properties of sensitiveA549 and resistant A549/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A549 cell and 0.194 for the resistant A549/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A549/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that altera-tions of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A549 cell membranes was looser than that of the A549/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A549/DDP cells was also lower than that of A549 cells. Taken together, it was proposed that the al-teration of membrane lipid biophysical state may be involved in the resistance of A549/DDP cells to cisplatin.

  1. Selenium-enriched durum wheat improves the nutritional profile of pasta without altering its organoleptic properties.

    Science.gov (United States)

    De Vita, Pasquale; Platani, Cristiano; Fragasso, Mariagiovanna; Ficco, Donatella Bianca Maria; Colecchia, Salvatore Antonio; Del Nobile, Matteo Alessandro; Padalino, Lucia; Di Gennaro, Spartaco; Petrozza, Angelo

    2017-01-01

    Two field experiments were conducted over three growing seasons (2006-07, 2008-09 and 2009-10) to evaluate Se-enriched pasta through foliar fertilization at various rates and timing of application on 4 durum wheat varieties. Our findings confirm the effectiveness of foliar Se fertilization to increase Se concentrations in durum wheat grain, even at high Se rates (120gSeha(-1)). Se fortification was significant across different genotypes, with greater Se accumulation in landraces ('Timilia') and obsolete varieties ('Cappelli'), with respect to modern varieties. The Se content in the grain was increased by up to 35-fold that of the untreated control. The Se concentration decreased during milling (11%), while processing and cooking of pasta did not show significant decreases. This biofortification stategy had no effects on grain quality parameters, except for reduced gluten index in the high-gluten variety PR22D89, as well as for the sensorial properties of the spaghetti.

  2. Diminished A-type potassium current and altered firing properties in presympathetic PVN neurones in renovascular hypertensive rats.

    Science.gov (United States)

    Sonner, Patrick M; Filosa, Jessica A; Stern, Javier E

    2008-03-15

    Accumulating evidence supports a contribution of the hypothalamic paraventricular nucleus (PVN) to sympathoexcitation and elevated blood pressure in renovascular hypertension. However, the underlying mechanisms resulting in altered neuronal function in hypertensive rats remain largely unknown. Here, we aimed to address whether the transient outward potassium current (I(A)) in identified rostral ventrolateral medulla (RVLM)-projecting PVN neurones is altered in hypertensive rats, and whether such changes affected single and repetitive action potential properties and associated changes in intracellular Ca(2+) levels. Patch-clamp recordings obtained from PVN-RVLM neurons showed a reduction in I(A) current magnitude and single channel conductance, and an enhanced steady-state current inactivation in hypertensive rats. Morphometric reconstructions of intracellularly labelled PVN-RVLM neurons showed a diminished dendritic surface area in hypertensive rats. Consistent with a diminished I(A) availability, action potentials in PVN-RVLM neurons in hypertensive rats were broader, decayed more slowly, and were less sensitive to the K(+) channel blocker 4-aminopyridine. Simultaneous patch clamp recordings and confocal Ca(2+) imaging demonstrated enhanced action potential-evoked intracellular Ca(2+) transients in hypertensive rats. Finally, spike broadening during repetitive firing discharge was enhanced in PVN-RVLM neurons from hypertensive rats. Altogether, our results indicate that diminished I(A) availability constitutes a contributing mechanism underlying aberrant central neuronal function in renovascular hypertension.

  3. Using deposition rate as a means to alter the properties of small molecule organic glasses for OLED applications

    Science.gov (United States)

    Kearns, Kenneth; Krzyskowski, Paige; Devereaux, Zachary

    2015-03-01

    Organic light emitting diode (OLED) devices rely on vapor-deposited, small molecule organic glasses. Recent work has shown that deposition condition plays a critical role in altering OLED device performance. Here it will be shown that the deposition rate alters the onset and fictive temperatures measured by differential scanning calorimetry (DSC) of the deposited glass. Glasses of the common hole transport materials NPD and TPD were prepared with onset temperatures 17 and 16 K higher, respectively, than the ordinary glass prepared by cooling the supercooled liquid. The thermal stability of glasses in functioning devices can be underestimated due to increases in onset temperature relative to Tg. The fictive temperatures for NPD and TPD were 32 and 27 K lower, respectively, than the Tg of the ordinary glass. These results are consistent with literature reports on other non-OLED glasses where enhanced surface mobility allowed for glasses with similar properties to be made. Ellipsometry studies on NPD showed that the fictive and onset temperatures were consistent with the DSC results. Additionally, the modeled birefringence of the as-deposited NPD glass was non-zero and quickly decreased upon heating above the onset temperature, which has implications for device performance. Formerly at Department of Chemistry, Saginaw Valley State University.

  4. Abnormal physiological properties and altered cell wall composition in Streptococcus pneumoniae grown in the presence of clavulanic acid.

    Science.gov (United States)

    Severin, A; Severina, E; Tomasz, A

    1997-01-01

    Subinhibitory concentrations of clavulanate caused premature induction of stationary-phase autolysis, sensitization to lysozyme, and reductions in the MICs of deoxycholate and penicillin for Streptococcus pneumoniae. In the range of clavulanate concentrations producing these effects, this beta-lactam compound was selectively bound to PBP 3. Cell walls isolated from pneumococci grown in the presence of clavulanate showed increased sensitivity to the hydrolytic action of purified pneumococcal autolysin in vitro. High-performance liquid chromatography analysis of the peptidoglycan isolated from the clavulanate-grown cells showed major qualitative and quantitative changes in stem peptide composition, the most striking feature of which was the accumulation of peptide species carrying intact D-alanyl-D-alanine residues at the carboxy termini. The altered biological and biochemical properties of the clavulanate-grown pneumococci appear to be the consequences of suppressed D,D-carboxypeptidase activity. PMID:9055983

  5. Current considerations concerning endodontically treated teeth: alteration of hard dental tissues and biomechanical properties following endodontic therapy.

    Science.gov (United States)

    Dimitriu, Bogdan; Vârlan, Constantin; Suciu, Ioana; Vârlan, Virginia; Bodnar, Dana

    2009-01-01

    The aim of this general article is to present an overview of the current knowledge about composition and structural changes and also about specific biomechanical alterations related to vitality loss or endodontic therapy. For a long time, these issues have been controversially approached from a clinical standpoint and are therefore still confusing for many practitioners. Vitality loss or endodontic procedures seem to induce only negligible changes in hard dental tissue moisture. Physico-chemical properties of dentin can be modified by some of the endodontic chemical products used for chemo-mechanical debridement. On the other hand, tooth biomechanical behavior is affected, since tooth strength is reduced proportionally to coronal tissue loss, due to either pre-existent carious/non-carious lesions or cavity acces preparation, besides restorative procedures. The related literature shows the lack of accepted clinical standards and consensus regarding the optimal way of approaching the specific tooth biomechanics following endodontic therapy.

  6. Dynamic Alterations in Microarchitecture, Mineralization and Mechanical Property of Subchondral Bone in Rat Medial Meniscal Tear Model of Osteoarthritis

    Institute of Scientific and Technical Information of China (English)

    De-Gang Yu; Shao-Bo Nie; Feng-Xiang Liu; Chuan-Long Wu; Bo Tian; Wen-Gang Wang; Xiao-Qing Wang

    2015-01-01

    Background:The properties of subchondral bone influence the integrity of articular cartilage in the pathogenesis of osteoarthritis (OA).However,the characteristics of subchondral bone alterations remain unresolved.The present study aimed to observe the dynamic alterations in the microarchitecture,mineralization,and mechanical properties of subchondral bone during the progression of OA.Methods:A medial meniscal tear (MMT) operation was performed in 128 adult Sprague Dawley rats to induce OA.At 2,4,8,and 12 weeks following the MMT operation,cartilage degeneration was evaluated using toluidine blue O staining,whereas changes in the microarchitecture indices and tissue mineral density (TMD),mineral-to-collagen ratio,and intrinsic mechanical properties of subchondral bone plates (BPs) and trabecular bones (Tbs) were measured using micro-computed tomography scanning,confocal Raman microspectroscopy and nanoindentation testing,respectively.Results:Cartilage degeneration occurred and worsened progressively from 2 to 12 weeks after OA induction.Microarchitecture analysis revealed that the subchondral bone shifted from bone resorption early (reduced trabecular BV/TV,trabecular number,connectivity density and trabecular thickness [Tb.Th],and increased trabecular spacing (Tb.Sp) at 2 and 4 weeks) to bone accretion late (increased BV/TV,Tb.Th and thickness of subchondral bone plate,and reduced Tb.Sp at 8 and 12 weeks).The TMD of both the BP and Tb displayed no significant changes at 2 and 4 weeks but decreased at 8 and 12 weeks.The mineral-to-collagen ratio showed a significant decrease from 4 weeks for the Tb and from 8 weeks for the BP after OA induction.Both the elastic modulus and hardness of the Tb showed a significant decrease from 4 weeks after OA induction.The BP showed a significant decrease in its elastic modulus from 8 weeks and its hardness from 4 weeks.Conclusion:The microarchitecture,mineralization and mechanical properties of subchondral bone changed in a time

  7. Alterations in cancer cell mechanical properties after fluid shear stress exposure: a micropipette aspiration study

    Directory of Open Access Journals (Sweden)

    Chivukula VK

    2015-01-01

    Full Text Available Venkat Keshav Chivukula,1 Benjamin L Krog,1,2 Jones T Nauseef,2 Michael D Henry,2 Sarah C Vigmostad1 1Department of Biomedical Engineering, 2Department of Molecular Physiology and Biophysics, Holden Comprehensive Cancer Center, University of Iowa, Seamans Center for the Engineering Arts and Sciences, Iowa City, IA, USA Abstract: Over 90% of cancer deaths result not from primary tumor development, but from metastatic tumors that arise after cancer cells circulate to distal sites via the circulatory system. While it is known that metastasis is an inefficient process, the effect of hemodynamic parameters such as fluid shear stress (FSS on the viability and efficacy of metastasis is not well understood. Recent work has shown that select cancer cells may be able to survive and possibly even adapt to FSS in vitro. The current research seeks to characterize the effect of FSS on the mechanical properties of suspended cancer cells in vitro. Nontransformed prostate epithelial cells (PrEC LH and transformed prostate cancer cells (PC-3 were used in this study. The Young's modulus was determined using micropipette aspiration. We examined cells in suspension but not exposed to FSS (unsheared and immediately after exposure to high (6,400 dyn/cm2 and low (510 dyn/cm2 FSS. The PrEC LH cells were ~140% stiffer than the PC-3 cells not exposed to FSS. Post-FSS exposure, there was an increase of ~77% in Young's modulus after exposure to high FSS and a ~47% increase in Young's modulus after exposure to low FSS for the PC-3 cells. There was no significant change in the Young's modulus of PrEC LH cells post-FSS exposure. Our findings indicate that cancer cells adapt to FSS, with an increased Young's modulus being one of the adaptive responses, and that this adaptation is specific only to PC-3 cells and is not seen in PrEC LH cells. Moreover, this adaptation appears to be graded in response to the magnitude of FSS experienced by the cancer cells. This is the first study

  8. Altered Biomechanical Properties of Gastrocnemius Tendons of Turkeys Infected with Turkey Arthritis Reovirus

    Directory of Open Access Journals (Sweden)

    Tamer A. Sharafeldin

    2016-01-01

    Full Text Available Turkey arthritis reovirus (TARV causes lameness and tenosynovitis in commercial turkeys and is often associated with gastrocnemius tendon rupture by the marketing age. This study was undertaken to characterize the biomechanical properties of tendons from reovirus-infected turkeys. One-week-old turkey poults were orally inoculated with O’Neil strain of TARV and observed for up to 16 weeks of age. Lameness was first observed at 8 weeks of age, which continued at 12 and 16 weeks. At 4, 8, 12, and 16 weeks of age, samples were collected from legs. Left intertarsal joint with adjacent gastrocnemius tendon was collected and processed for histological examination. The right gastrocnemius tendon’s tensile strength and elasticity modulus were analyzed by stressing each tendon to the point of rupture. At 16 weeks of age, gastrocnemius tendons of TARV-infected turkeys showed significantly reduced (P<0.05 tensile strength and modulus of elasticity as compared to those of noninfected control turkeys. Gastrocnemius tendons revealed lymphocytic tendinitis/tenosynovitis beginning at 4 weeks of age, continuing through 8 and 12 weeks, and progressing to fibrosis from 12 to 16 weeks of age. We propose that tendon fibrosis is one of the key features contributing to reduction in tensile strength and elasticity of gastrocnemius tendons in TARV-infected turkeys.

  9. Corticosteroid administration alters the mechanical properties of isolated collagen fascicles in rat-tail tendon.

    Science.gov (United States)

    Haraldsson, B T; Aagaard, P; Crafoord-Larsen, D; Kjaer, M; Magnusson, S P

    2009-10-01

    Overload tendon injuries are frequent in recreational and elite sports. The optimal treatment strategy remains unknown, but local administration of corticosteroids is one common treatment option. The direct effects of the corticosteroid administration on the tissue are not fully understood. The present study examined the biomechanical effects of intratendinous corticosteroid injections on healthy rat-tail tendon collagen fascicles. A total of 24 Wistar male rats were divided into (A) a corticosteroid group where the animals were injected in the tail tendon with methylprednisolone acetate, 1.0 mL of 40 mg/mL mixed with 1.0 mL 9% saline (n=12), and (B) a control group that was injected with 9% saline (n=12). Three days after the injections, the animals were sacrificed and single individual collagen fascicles were collected and underwent displacement to failure. Corticosteroid administration significantly reduced tensile fascicle yield strength by 16% and Young's modulus by 14% compared with sham treatment (10.5+/-0.8 vs 12.4+/-0.5 MPa, P< or =0.05, and 537+/-27 vs 641+/-30 MPa, P<0.05, respectively), while the strain properties were unaffected. Peak stress was similar between the two groups. There was no difference in fascicle diameter between the two groups.

  10. Designed synthesis and chiroptical properties of regioregular poly(p-phenyleneethynylene-alter-m-phenyleneethynylene) bearing (-)-trans-rnyrtanoxyl side groups

    Institute of Scientific and Technical Information of China (English)

    Xiangfeng LI; Chusheng LI; Jiang LU; Hui LIANG

    2009-01-01

    Two regioregular poly(p-phenyleneethyny-lene-alter-m-phenyleneethynylene)s bearing (-)-trans-myrtanoxyl side groups with different substitution patterns were designed and synthesized, e.g. Myr-PMPE-1 and Myr-PMPE-2. In Myr-PMPE-1, the side chiral groups are distributed uniformly along the backbone. In Myr-PMPE-2, the distribution of the side chiral groups is alternatively crowded and loose. Both of these two polymers show no CD signal in solutions because of their good solubility. The investigations of chiroptical properties of these two polymers were carried out in the form of spin-coated films. The films were annealed above the glass temperature of the corresponding polymer, and the effects of annealing temperature and time on the properties of the films were investigated by UV-Vis absorption, fluorescence and circular dichroism spectra. The results show that annealing treatment had no significant effect on the properties of Myr-PMPE-1, including UV-Vis absorption, fluorescence and optical activity. The maximum absolute value of dissymmetry factor (|gmax|) was 1.62 × 10-4. On the other hand, annealing treatment significantly affected the proper-ties of Myr-PMPE-2. Without annealing or being annealed below 100℃, Myr-PMPE-2 films show almost no Cotton effect. In contrast, when annealed above 120℃, the absorption and emission of Myr-PMPE-2 films slightly red shifted with increasing annealing temperature and annealing time. Most importantly, the intensity of CD signals increased significantly and the optical activity of Myr-PMPE-2 films markedly increased. After annealing at 140℃ for 4h, the |gmax| of Myr-PMPE-2 films was increased up to 3.07 x 10 3, about one order of magnitude higher than that of Myr-PMPE-1 films.

  11. Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

    Directory of Open Access Journals (Sweden)

    Matthew S Barnabei

    Full Text Available Duchenne muscular dystrophy (DMD is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

  12. Physical Properties of Blood Are Altered in Young and Lean Women with Polycystic Ovary Syndrome

    Science.gov (United States)

    Simmonds, Michael J.; Milne, Nikki; Ong, Kee; Brotherton, Emily; McNamee, Antony P.; Horobin, Jarod; Sabapathy, Surendran

    2016-01-01

    Classic features of polycystic ovary syndrome (PCOS) include derangement of metabolic and cardiovascular health, and vascular dysfunction is commonly reported. These comorbidities indicate impaired blood flow; however, other than limited reports of increased plasma viscosity, surprisingly little is known regarding the physical properties of blood in PCOS. We aimed to investigate whether haemorheology was impaired in women with PCOS. We thus measured a comprehensive haemorheological profile, in a case-control design, of lean women with PCOS and age-matched healthy controls. A clinical examination determined similar cardiovascular risk for the two groups. Whole blood and plasma viscosity was measured using a cone-plate viscometer. The magnitude and rate of red blood cell (RBC) aggregation was determined using a light-transmission aggregometer, and the degree of RBC deformability was measured via laser-diffraction ektacytometry. Plasma viscosity was significantly increased in women with PCOS. Blood viscosity was also increased for PCOS at lower-to-moderate shear rates in both native and standardised haematocrit samples. The magnitude of RBC aggregation–a primary determinant of low-shear blood viscosity–was significantly increased in PCOS at native and 0.4 L·L-1 haematocrit. No difference was detected between PCOS and CON groups for RBC deformability measurements. A novel measure indicating the effectiveness of oxygen transport by RBC (i.e., the haematocrit-to-viscosity ratio; HVR) was decreased at all shear rates in women with PCOS. In a group of young and lean women with PCOS with an unremarkable cardiovascular risk profile based on clinical data, significant haemorheological impairment was observed. The degree of haemorheological derangement observed in the present study reflects that of overt chronic disease, and provides an avenue for future therapeutic intervention in PCOS. PMID:27902766

  13. Physical and chemical modifications of surface properties lead to alterations in osteoblast behavior

    Science.gov (United States)

    Dorst, Kathryn Elizabeth

    Proper formation of the bone extracellular matrix (ECM), or osteoid, depends on the surface properties of pre-existing tissue and the aqueous chemical environment. Both of these factors greatly influence osteoblast migration, cytoskeletal organization, and calcium nodule production, important aspects when considering the biocompatibility of bone implants. By perturbing the physical and/or chemical micro-environment, it may be possible to elucidate effects on cellular function. To examine these factors, murine pre-osteoblasts (MC3T3-E1 subclones 4 and 24) were seeded on polydimethylsiloxane (PDMS) substrates containing "wide" micro-patterned ridges (20 mum width, 30 mum pitch, & 2 mum height), "narrow" micro-patterned ridges (2 mum width, 10 mum pitch, 2 mum height), no patterns (flat PDMS), and standard tissue culture (TC) polystyrene as a control. Zinc concentration was adjusted to mimic deficient (0.23 muM), serum-level (3.6 muM), and zinc-rich (50 muM) conditions. It was found that cells exhibited distinct anisotropic migration in serum-level zinc and zinc-deficient media on the wide PDMS patterns, however this was disrupted under zinc-rich conditions. Production of differentiation effectors, activated metalloproteinase-2 (MMP-2) and transforming growth factor - beta 1 (TGF-beta1), was increased with the addition of exogenous zinc. Early stage differentiation, via alkaline phosphatase, was modified by zinc levels on patterned polydimethylsiloxane (PDMS) surfaces, but not on flat PDMS or tissue culture polystyrene (TC). Late stage differentiation, visualized through calcium phosphate nodules, was markedly different at various zinc levels when the cells were cultured on TC substrates. This susceptibility to zinc content can lead to differences in bone mineral production on certain substrates if osteoblasts are not able to maintain and remodel bone effectively, a process vital to successful biomaterial integration.

  14. Muscle cramp in Machado-Joseph disease: altered motor axonal excitability properties and mexiletine treatment.

    Science.gov (United States)

    Kanai, Kazuaki; Kuwabara, Satoshi; Arai, Kimihito; Sung, Jia-Ying; Ogawara, Kazue; Hattori, Takamichi

    2003-04-01

    Machado-Joseph disease is one of the most common hereditary spinocerebellar degenerative disorders with a wide range of clinical manifestations. Pathology studies have shown mild to moderate loss of anterior horn cells and, in terms of spinal pathology, Machado-Joseph disease is regarded as a type of lower motoneuron disease. Muscle cramps are often associated with lower motoneuron disorders, but features of cramps in Machado-Joseph disease patients have never been studied. We investigated the incidence and nature of muscle cramps in Machado-Joseph disease patients, the excitability properties of motor axons [strength-duration time constant (tau(SD)), threshold electrotonus, refractoriness and supernormality] using threshold tracking and the effects of mexiletine hydrochloride on those cramps. Of 20 consecutive patients, 16 (80%) had frequent, severe muscle cramps in the legs, trunk or arms that disturbed their daily activities. The frequency of pathological muscle cramps was similar to that for patients with amyotrophic lateral sclerosis (68%) and higher than those for patients with spinal muscular atrophy (33%) or peripheral axonal neuropathy (24%). Threshold-tracking studies showed that tau(SD), which in part reflects Na(+) conductance at the resting membrane potential, was significantly greater in the Machado-Joseph disease patients than in normal subjects; severe muscle cramps were associated with a longer tau(SD). Threshold electrotonus, refractoriness and supernormality were not significantly different between Machado-Joseph disease patients and normal subjects. Eight Machado-Joseph disease patients with severe cramps, who received mexiletine treatment, experienced nearly complete relief with a partial normalization of tau(SD) (P = 0.08). Muscle cramps are a very frequent and disabling factor in Machado-Joseph disease. Pathological muscle cramps responded well to mexiletine treatment, and this is consistent with the hypothesis that they are caused by an

  15. Characterization and petrophysical properties of hydrothemally altered lacustrine volcanistic rock in Geyser Valley (Kamchatka) and its transformation by weathering

    Science.gov (United States)

    Gvozdeva, Irina; Zerkal, Oleg; Samarin, Evgeny

    2013-04-01

    content of clay minerals (to 90%), decreasing the content of zeolites (not to exceed 10%). Quartz and plagioclase form sans fraction. Physical and mechanical properties vary widely: the density of the soil increases slightly up to 1,57-1,59 g/cm3 for sands, 1,2-1,79 g/cm3 for clays, porosity of 51-52% and 49-78% respectively, moisture 22-23% and 43-98/ Clays are in a state of semi-solid to fluid. The high content of smectite determines high plastic properties. Plasticity Index varies widely from 11 to 57. Cohesion and the internal friction angle obtained from shear tests also change widely. For clayey sand grip reaches 137 kPa, internal friction angle - 17 degrees. In clay grip ranges from 13 kPa to 120 kPa, and the internal friction angle - from 11 degrees to 31 degrees. Large variation of properties of the investigated soils is explained by the inhomogeneity of volcano-sedimentary formations both vertically and laterally, varying degrees of hydrothermal alteration and of weathering, fracturing and cracks filling The obtained datas can adequately characterize the volcanic-lacustrine sediments in the valley of the Geysernaya river and use them in calculations of slope stability and for and geological mapping.

  16. Structure alteration and immunological properties of {sup 60}Co gamma rays irradiated bothropstoxin-I

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, Janaina A.; Yonamine, Camila Myiagui; Caproni, Priscila; Casare, Murilo; Spencer, Patrick Jack; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail: janabap@gmail.com; Andrade Junior, Heitor Franco de; Vieira, Daniel Perez; Galisteo Junior, Andres Jimenez [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Instituto de Medicina Tropical de Sao Paulo, SP (Brazil). Lab. de Protozoologia

    2007-07-01

    About 20000 ophidic accidents are registered every year in Brazil. Serum therapy with equine antisera is the only efficient treatment. The venoms employed for immunization are fairly toxic and some venoms present low immunogenicity. Thus, the obtention of modified antigens with lower toxicity and preserved or improved immunogenicity would be useful. These toxins, when submitted to gamma radiation, in aqueous solution, present structural modifications. This occurs due to reactions with the radiolysis products of water. Some scavenger substances, such as NaNO{sub 3} and t-butanol, remove selectively the water radiolysis products. Ionizing radiation has proven to be a powerful tool to attenuate snake venoms toxicity without affecting and even increasing their immunogenic properties. However, the immune mechanisms involved in recognition, processing and presentation of irradiated antigens are yet unclear. In the present work, we investigated the immunological behavior of bothropstoxin-I (Bthx-1), before and after irradiation, in the presence of selective scavengers. Isogenic mice were immunized with either the native or the irradiated toxin, either with or without scavengers. After three immunizations, serum samples were collected and the antibody titers and isotypes were determined by Enzyme Linked Immuno Sorbent Assay. The antigenic characterization of native and irradiated bothropstoxin-I was performed by Western blot. The detection of expression of murine cytokines (IFN-{gamma} and IL-10) was analyzed by RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). According to our data, irradiation process has promoted structural modifications in the toxin, characterized by higher molecular weight forms of the protein (aggregates and oligomers). Our data also indicate that irradiated toxins, alone or in the presence of NaNO{sub 3}, an aqueous electron scavenger, were immunogenic and the antibodies elicited by them were able to recognize the native toxin. On the other

  17. The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Geppert, Mark; Petters, Charlotte [University of Bremen, Centre for Biomolecular Interactions Bremen (Germany); Thiel, Karsten [Fraunhofer Institute for Manufacturing Technology and Advanced Materials (Germany); Dringen, Ralf, E-mail: ralf.dringen@uni-bremen.de [University of Bremen, Centre for Biomolecular Interactions Bremen (Germany)

    2013-01-15

    Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood-brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 {+-} 2 to 107 {+-} 6 nm and the zeta-potential of the particles from -22 {+-} 5 to -9 {+-} 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

  18. Exopolymer alteration of physical properties of sea ice and implications for ice habitability and biogeochemistry in a warmer Arctic.

    Science.gov (United States)

    Krembs, Christopher; Eicken, Hajo; Deming, Jody W

    2011-03-01

    The physical properties of Arctic sea ice determine its habitability. Whether ice-dwelling organisms can change those properties has rarely been addressed. Following discovery that sea ice contains an abundance of gelatinous extracellular polymeric substances (EPS), we examined the effects of algal EPS on the microstructure and salt retention of ice grown from saline solutions containing EPS from a culture of the sea-ice diatom, Melosira arctica. We also experimented with xanthan gum and with EPS from a culture of the cold-adapted bacterium Colwellia psychrerythraea strain 34H. Quantitative microscopic analyses of the artificial ice containing Melosira EPS revealed convoluted ice-pore morphologies of high fractal dimension, mimicking features found in EPS-rich coastal sea ice, whereas EPS-free (control) ice featured much simpler pore geometries. A heat-sensitive glycoprotein fraction of Melosira EPS accounted for complex pore morphologies. Although all tested forms of EPS increased bulk ice salinity (by 11-59%) above the controls, ice containing native Melosira EPS retained the most salt. EPS effects on ice and pore microstructure improve sea ice habitability, survivability, and potential for increased primary productivity, even as they may alter the persistence and biogeochemical imprint of sea ice on the surface ocean in a warming climate.

  19. RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site

    Directory of Open Access Journals (Sweden)

    Feng Cheng

    2017-01-01

    Full Text Available RNA-binding proteins (RBPs and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1, is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (p=0.04. Patients with higher Dnd1 expression level had longer overall survival (p=0.0014 by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3′UTR, the stability of Bim-5′UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3′UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3′UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

  20. RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site.

    Science.gov (United States)

    Cheng, Feng; Pan, Ying; Lu, Yi-Min; Zhu, Lei; Chen, Shuzheng

    2017-01-01

    RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (p = 0.04). Patients with higher Dnd1 expression level had longer overall survival (p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3'UTR, the stability of Bim-5'UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3'UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3'UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

  1. Structural analyses of the CRISPR protein Csc2 reveal the RNA-binding interface of the type I-D Cas7 family.

    Science.gov (United States)

    Hrle, Ajla; Maier, Lisa-Katharina; Sharma, Kundan; Ebert, Judith; Basquin, Claire; Urlaub, Henning; Marchfelder, Anita; Conti, Elena

    2014-01-01

    Upon pathogen invasion, bacteria and archaea activate an RNA-interference-like mechanism termed CRISPR (clustered regularly interspaced short palindromic repeats). A large family of Cas (CRISPR-associated) proteins mediates the different stages of this sophisticated immune response. Bioinformatic studies have classified the Cas proteins into families, according to their sequences and respective functions. These range from the insertion of the foreign genetic elements into the host genome to the activation of the interference machinery as well as target degradation upon attack. Cas7 family proteins are central to the type I and type III interference machineries as they constitute the backbone of the large interference complexes. Here we report the crystal structure of Thermofilum pendens Csc2, a Cas7 family protein of type I-D. We found that Csc2 forms a core RRM-like domain, flanked by three peripheral insertion domains: a lid domain, a Zinc-binding domain and a helical domain. Comparison with other Cas7 family proteins reveals a set of similar structural features both in the core and in the peripheral domains, despite the absence of significant sequence similarity. T. pendens Csc2 binds single-stranded RNA in vitro in a sequence-independent manner. Using a crosslinking - mass-spectrometry approach, we mapped the RNA-binding surface to a positively charged surface patch on T. pendens Csc2. Thus our analysis of the key structural and functional features of T. pendens Csc2 highlights recurring themes and evolutionary relationships in type I and type III Cas proteins.

  2. AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins

    Indian Academy of Sciences (India)

    ADWAITA PRASAD PARIDA; AMRAPALI SHARMA; ARUN KUMAR SHARMA

    2017-03-01

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome fromtransposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG bindingdomain proteins are members of a class of proteins that bind tomethylated DNA. The Arabidopsis thaliana genome encodes13 methyl CpG binding domain (MBD) proteins, but themolecular/biological functions of most of these proteins are still notclear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNAbinding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40Sribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physicallyinteracts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component ofthe RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci,pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved inRNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 alsointeracts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

  3. Trypanosoma brucei RNA binding proteins p34 and p37 mediate NOPP44/46 cellular localization via the exportin 1 nuclear export pathway.

    Science.gov (United States)

    Hellman, Kristina; Prohaska, Kimberly; Williams, Noreen

    2007-12-01

    We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.

  4. RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site

    Science.gov (United States)

    Cheng, Feng; Pan, Ying; Lu, Yi-Min; Zhu, Lei

    2017-01-01

    RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (p = 0.04). Patients with higher Dnd1 expression level had longer overall survival (p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3′UTR, the stability of Bim-5′UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3′UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3′UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

  5. Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.

    Science.gov (United States)

    Murray, Rebecca D; Merchant, Michael L; Hardin, Ericka; Clark, Barbara; Khundmiri, Syed J; Lederer, Eleanor D

    2016-02-01

    Parathyroid hormone (PTH) is a key regulator of the expression and function of the type IIa sodium-phosphate cotransporter (Npt2a), the protein responsible for regulated renal phosphate reabsorption. We previously showed that PTH induces rapid decay of Npt2a mRNA through posttranscriptional mechanisms. We hypothesized that PTH-induced changes in RNA-binding protein (RBP) activity mediate the degradation of Npt2a mRNA. To address this aim, we treated opossum kidney (OK) cells, a PTH-sensitive proximal tubule cell culture model, with 100 nM PTH for 30 min and 2 h, followed by mass spectrometry characterization of the PTH-stimulated phosphoproteome. We identified 1,182 proteins differentially phosphorylated in response to PTH, including 68 RBPs. Preliminary analysis identified a phospho-RBP, hnRNPK-homology-type-splicing regulatory protein (KSRP), with predicted binding sites for the 3'-untranslated region (UTR) of Npt2a mRNA. Western blot analysis confirmed expression of KSRP in OK cells and showed PTH-dependent translocation to the nucleus. Immunoprecipitation of KSRP from control and PTH-treated cells followed by RNA isolation and RT-quantitative PCR analysis identified Npt2a mRNA from both control and PTH-treated KSRP pulldowns. Knockdown of KSRP followed by PTH treatment showed that KSRP is required for mediating PTH-stimulated reduction in sodium/hydrogen exchanger 3 mRNA, but not Npt2a mRNA. We conclude that 1) PTH is a major regulator of both transcription and translation, and 2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.

  6. The TRIM-NHL protein LIN-41 and the OMA RNA-binding proteins antagonistically control the prophase-to-metaphase transition and growth of Caenorhabditis elegans oocytes.

    Science.gov (United States)

    Spike, Caroline A; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-12-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation.

  7. RNA-binding protein HuD reduces triglyceride production in pancreatic β cells by enhancing the expression of insulin-induced gene 1.

    Science.gov (United States)

    Kim, Chongtae; Lee, Heejin; Kang, Hoin; Shin, Jung Jae; Tak, Hyosun; Kim, Wook; Gorospe, Myriam; Lee, Eun Kyung

    2016-04-01

    Although triglyceride (TG) accumulation in the pancreas leads to β-cell dysfunction and raises the chance to develop metabolic disorders such as type 2 diabetes (T2DM), the molecular mechanisms whereby intracellular TG levels are regulated in pancreatic β cells have not been fully elucidated. Here, we present evidence that the RNA-binding protein HuD regulates TG production in pancreatic β cells. Mouse insulinoma βTC6 cells stably expressing a small hairpin RNA targeting HuD (shHuD) (βTC6-shHuD) contained higher TG levels compared to control cells. Moreover, downregulation of HuD resulted in a decrease in insulin-induced gene 1 (INSIG1) levels but not in the levels of sterol regulatory element-binding protein 1c (SREBP1c), a key transcription factor for lipid production. We identified Insig1 mRNA as a direct target of HuD by using ribonucleoprotein immunoprecipitation (RIP) and biotin pulldown analyses. By associating with the 3'-untranslated region (3'UTR) of Insig1 mRNA, HuD promoted INSIG1 translation; accordingly, HuD downregulation reduced while ectopic HuD expression increased INSIG1 levels. We further observed that HuD downregulation facilitated the nuclear localization of SREBP1c, thereby increasing the transcriptional activity of SREBP1c and the expression of target genes involved in lipogenesis; likewise, we observed lower INSIG1 levels in the pancreatic islets of HuD-null mice. Taken together, our results indicate that HuD functions as a novel repressor of lipid synthesis in pancreatic β cells.

  8. Double-stranded RNA-binding protein DRB3 negatively regulates anthocyanin biosynthesis by modulating PAP1 expression in Arabidopsis thaliana.

    Science.gov (United States)

    Sawano, Hikaru; Matsuzaki, Takuma; Usui, Tomoyuki; Tabara, Midori; Fukudome, Akihito; Kanaya, Akihiro; Tanoue, Daichi; Hiraguri, Akihiro; Horiguchi, Gorou; Ohtani, Misato; Demura, Taku; Kozaki, Toshinori; Ishii, Kazuo; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2017-01-01

    The model plant Arabidopsis thaliana has five double-stranded RNA-binding proteins (DRB1-DRB5), two of which, DRB1 and DRB4, are well characterized. In contrast, the functions of DRB2, DRB3 and DRB5 have yet to be elucidated. In this study, we tried to uncover their functions using drb mutants and DRB-over-expressed lines. In over-expressed lines of all five DRB genes, the over-expression of DRB2 or DRB3 (DRB2ox or DRB3ox) conferred a downward-curled leaf phenotype, but the expression profiles of ten small RNAs were similar to that of the wild-type (WT) plant. Phenotypes were examined in response to abiotic stresses. Both DRB2ox and DRB3ox plants exhibited salt-tolerance. When these plants were exposed to cold stress, drb2 and drb3 over-accumulated anthocyanin but DRB2ox and DRB3ox did not. Therefore, the over-expression of DRB2 or DRB3 had pleiotropic effects on host plants. Microarray and deep-sequencing analyses indicated that several genes encoding key enzymes for anthocyanin biosynthesis, including chalcone synthase (CHS), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS), were down-regulated in DRB3ox plants. When DRB3ox was crossed with the pap1-D line, which is an activation-tagged transgenic line that over-expresses the key transcription factor PAP1 (Production of anthocyanin pigmentation1) for anthocyanin biosynthesis, over-expression of DRB3 suppressed the expression of PAP1, CHS, DFR and ANS genes. DRB3 negatively regulates anthocyanin biosynthesis by modulating the level of PAP1 transcript. Since two different small RNAs regulate PAP1 gene expression, a possible function of DRB3 for small RNA biogenesis is discussed.

  9. Increase of the RNA-binding protein HuD and posttranscriptional up-regulation of the GAP-43 gene during spatial memory

    Science.gov (United States)

    Pascale, Alessia; Gusev, Pavel A.; Amadio, Marialaura; Dottorini, Tania; Govoni, Stefano; Alkon, Daniel L.; Quattrone, Alessandro

    2004-01-01

    Neuronal ELAV-like proteins (HuB, HuC, and HuD) are highly conserved RNA-binding proteins able to selectively associate with the 3′ UTR of a subset of target mRNAs and increase their cytoplasmic stability and rate of translation. We previously demonstrated the involvement of these proteins in learning, reporting that they undergo a sustained up-regulation in the hippocampus of mice trained in a spatial discrimination task. Here, we extend this finding, showing that a similar up-regulation occurs in the hippocampus of rats trained in another spatial learning paradigm, the Morris water maze. HuD, a strictly neuron-specific ELAV-like protein, is shown to increase after learning, with a preferential binding to the cytoskeletal fraction. HuD up-regulation is associated with an enhancement of GAP-43 mRNA and protein levels, with an apparently increased HuD colocalization with the GAP-43 mRNA and an increased association of neuronal ELAV-like proteins with the GAP-43 mRNA. These learning-dependent biochemical events appear to be spatiotemporally controlled, because they do not occur in another brain region involved in learning, the retrosplenial cortex, and at the level of protein expression they show extinction 1 month after training despite memory retention. By contrast, HuD mRNA levels still remain increased after 1 month in the CA1 region. This persistence may have implications for long-term memory recall. PMID:14745023

  10. Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

    Science.gov (United States)

    Graifer, D M; Babkina, G T; Matasova, N B; Vladimirov, S N; Karpova, G G; Vlassov, V V

    1989-07-01

    A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

  11. An RNA-binding complex involved in ribosome biogenesis contains a protein with homology to tRNA CCA-adding enzyme.

    Directory of Open Access Journals (Sweden)

    Jinzhong Lin

    2013-10-01

    Full Text Available A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA-protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.

  12. A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA.

    Science.gov (United States)

    Cibois, Marie; Gautier-Courteille, Carole; Vallée, Audrey; Paillard, Luc

    2010-01-01

    Targeted inactivations of RNA-binding proteins (RNA-BPs) can lead to huge phenotypical defects. These defects are due to the deregulation of certain mRNAs. However, we generally do not know, among the hundreds of mRNAs that are normally controlled by one RNA-BP, which are responsible for the observed phenotypes. Here, we designed an antisense oligonucleotide ("target protector") that masks the binding site of the RNA-BP CUG-binding protein 1 (CUGBP1) on the mRNA Suppressor of Hairless [Su(H)] that encodes a key player of Notch signaling. We showed that injecting this oligonucleotide into Xenopus embryos specifically inhibited the binding of CUGBP1 to the mRNA. This caused the derepression of Su(H) mRNA, the overexpression of Su(H) protein, and a phenotypic defect, loss of somitic segmentation, similar to that caused by a knockdown of CUGBP1. To demonstrate a causal relationship between Su(H) derepression and the segmentation defects, a rescue experiment was designed. Embryonic development was restored when the translation of Su(H) mRNA was re-repressed and the level of Su(H) protein was reduced to a normal level. This "target protector and rescue assay" demonstrates that the phenotypic defects associated with CUGBP1 inactivation in Xenopus are essentially due to the deregulation of Su(H) mRNA. Similar approaches may be largely used to uncover the links between the phenotype caused by the inactivation of an RNA-BP and the identity of the RNAs associated with that protein.

  13. rs2735383, located at a microRNA binding site in the 3’UTR of NBS1, is not associated with breast cancer risk

    Science.gov (United States)

    Liu, Jingjing; Lončar, Ivona; Collée, J. Margriet; Bolla, Manjeet K.; Dennis, Joe; Michailidou, Kyriaki; Wang, Qin; Andrulis, Irene L.; Barile, Monica; Beckmann, Matthias W.; Behrens, Sabine; Benitez, Javier; Blomqvist, Carl; Boeckx, Bram; Bogdanova, Natalia V.; Bojesen, Stig E.; Brauch, Hiltrud; Brennan, Paul; Brenner, Hermann; Broeks, Annegien; Burwinkel, Barbara; Chang-Claude, Jenny; Chen, Shou-Tung; Chenevix-Trench, Georgia; Cheng, Ching Y.; Choi, Ji-Yeob; Couch, Fergus J.; Cox, Angela; Cross, Simon S.; Cuk, Katarina; Czene, Kamila; Dörk, Thilo; dos-Santos-Silva, Isabel; Fasching, Peter A.; Figueroa, Jonine; Flyger, Henrik; García-Closas, Montserrat; Giles, Graham G.; Glendon, Gord; Goldberg, Mark S.; González-Neira, Anna; Guénel, Pascal; Haiman, Christopher A.; Hamann, Ute; Hart, Steven N.; Hartman, Mikael; Hatse, Sigrid; Hopper, John L.; Ito, Hidemi; Jakubowska, Anna; Kabisch, Maria; Kang, Daehee; Kosma, Veli-Matti; Kristensen, Vessela N.; Le Marchand, Loic; Lee, Eunjung; Li, Jingmei; Lophatananon, Artitaya; Jan Lubinski; Mannermaa, Arto; Matsuo, Keitaro; Milne, Roger L.; Sahlberg, Kristine K.; Ottestad, Lars; Kåresen, Rolf; Langerød, Anita; Schlichting, Ellen; Holmen, Marit Muri; Sauer, Toril; Haakensen, Vilde; Engebråten, Olav; Naume, Bjørn; Kiserud, Cecile E.; Reinertsen, Kristin V.; Helland, åslaug; Riis, Margit; Bukholm, Ida; Lønning, Per Eystein; Børresen-Dale, Anne-Lise; Grenaker Alnæs, Grethe I.; Neuhausen, Susan L.; Nevanlinna, Heli; Orr, Nick; Perez, Jose I. A.; Peto, Julian; Putti, Thomas C.; Pylkäs, Katri; Radice, Paolo; Sangrajrang, Suleeporn; Sawyer, Elinor J.; Schmidt, Marjanka K.; Schneeweiss, Andreas; Shen, Chen-Yang; Shrubsole, Martha J.; Shu, Xiao-Ou; Simard, Jacques; Southey, Melissa C.; Swerdlow, Anthony; Teo, Soo H.; Tessier, Daniel C.; Thanasitthichai, Somchai; Tomlinson, Ian; Torres, Diana; Truong, Thérèse; Tseng, Chiu-Chen; Vachon, Celine; Winqvist, Robert; Wu, Anna H.; Yannoukakos, Drakoulis; Zheng, Wei; Hall, Per; Dunning, Alison M.; Easton, Douglas F.; Hooning, Maartje J.; van den Ouweland, Ans M. W.; Martens, John W. M.; Hollestelle, Antoinette

    2016-01-01

    NBS1, also known as NBN, plays an important role in maintaining genomic stability. Interestingly, rs2735383 G > C, located in a microRNA binding site in the 3′-untranslated region (UTR) of NBS1, was shown to be associated with increased susceptibility to lung and colorectal cancer. However, the relation between rs2735383 and susceptibility to breast cancer is not yet clear. Therefore, we genotyped rs2735383 in 1,170 familial non-BRCA1/2 breast cancer cases and 1,077 controls using PCR-based restriction fragment length polymorphism (RFLP-PCR) analysis, but found no association between rs2735383CC and breast cancer risk (OR = 1.214, 95% CI = 0.936–1.574, P = 0.144). Because we could not exclude a small effect size due to a limited sample size, we further analyzed imputed rs2735383 genotypes (r2 > 0.999) of 47,640 breast cancer cases and 46,656 controls from the Breast Cancer Association Consortium (BCAC). However, rs2735383CC was not associated with overall breast cancer risk in European (OR = 1.014, 95% CI = 0.969–1.060, P = 0.556) nor in Asian women (OR = 0.998, 95% CI = 0.905–1.100, P = 0.961). Subgroup analyses by age, age at menarche, age at menopause, menopausal status, number of pregnancies, breast feeding, family history and receptor status also did not reveal a significant association. This study therefore does not support the involvement of the genotype at NBS1 rs2735383 in breast cancer susceptibility. PMID:27845421

  14. RNA-binding motif protein 5 inhibits the proliferation of cigarette smoke-transformed BEAS-2B cells through cell cycle arrest and apoptosis.

    Science.gov (United States)

    Lv, Xue-Jiao; Du, Yan-Wei; Hao, Yu-Qiu; Su, Zhen-Zhong; Zhang, Lin; Zhao, Li-Jing; Zhang, Jie

    2016-04-01

    Cigarette smoking has been shown to be the most significant risk factor for lung cancer. Recent studies have also indicated that RNA-binding motif protein 5 (RBM5) can modulate apoptosis and suppress tumor growth. The present study focused on the role of RBM5 in the regulation of cigarette smoke extract (CSE)-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. Herein, we exposed normal BEAS-2B cells for 8 days to varying concentrations of CSE or dimethylsulfoxide (DMSO), followed by a recovery period of 2 weeks. Next, the RBM5 protein was overexpressed in these transformed BEAS-2B cells though lentiviral infection. Later, the morphological changes, cell proliferation, cell cycle, apoptosis, invasion and migration were assessed. In addition, we analyzed the role of RBM5 in xenograft growth. The expression of RBM5 along with the genes related to cell cycle regulation, apoptosis and invasion were also examined. Finally, our results revealed that BEAS-2B cells exposed to 100 µg/ml CSE acquired phenotypic changes and formed tumors in nude mice, indicative of their cancerous transformation and had reduced RBM5 expression. Subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest, triggered apoptosis and inhibited their invasion and migration, including xenograft growth. Thus, we established an in vitro model of CSE-induced cancerous transformation and concluded that RBM5 overexpression inhibited the growth of these transformed cells through cell cycle arrest and induction of apoptosis. Therefore, our study suggests the importance of RBM5 in the pathogenesis of smoking-related cancer.

  15. DNA Mutagenic Activity and Capacity for HIV-1 Restriction of the Cytidine Deaminase APOBEC3G Depends on Whether DNA or RNA Binds to Tyrosine 315.

    Science.gov (United States)

    Polevoda, Bogdan; Joseph, Rebecca; Friedman, Alan E; Bennett, Ryan P; Greiner, Rebecca; De Zoysa, Thareendra; Stewart, Ryan A; Smith, Harold C

    2017-04-05

    APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. RNA and ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C-terminus of A3G to its N-terminus. We show here that the A3G tyrosines 181 and 315 directly cross-link ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an E. coli DNA mutator reporter, while Y181A and Y182A mutants retained ~50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Y315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Y315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity.

  16. Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells

    Science.gov (United States)

    Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Seuningen, Isabelle Van; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

    2017-01-01

    Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally. PMID:28262838

  17. Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

    Science.gov (United States)

    Someya, Tatsuhiko; Baba, Seiki; Fujimoto, Mai; Kawai, Gota; Kumasaka, Takashi; Nakamura, Kouji

    2012-01-01

    Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA. PMID:22053080

  18. Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant.

    Directory of Open Access Journals (Sweden)

    Liang Qiao

    Full Text Available Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln silkworm strain as a model. Injection of β-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant and Dazao (wild-type silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.

  19. Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans.

    Science.gov (United States)

    Wu, Ye; Arai, Amy C; Rumbaugh, Gavin; Srivastava, Anand K; Turner, Gillian; Hayashi, Takashi; Suzuki, Erika; Jiang, Yuwu; Zhang, Lilei; Rodriguez, Jayson; Boyle, Jackie; Tarpey, Patrick; Raymond, F Lucy; Nevelsteen, Joke; Froyen, Guy; Stratton, Mike; Futreal, Andy; Gecz, Jozef; Stevenson, Roger; Schwartz, Charles E; Valle, David; Huganir, Richard L; Wang, Tao

    2007-11-13

    Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans.

  20. Long-term reactive nitrogen loading alters soil carbon and microbial community properties in a subalpine forest ecosystem

    Science.gov (United States)

    Boot, Claudia M; Hall, Ed K.; Denef, Karolien; Baron, Jill S.

    2016-01-01

    Elevated nitrogen (N) deposition due to increased fossil fuel combustion and agricultural practices has altered global carbon (C) cycling. Additions of reactive N to N-limited environments are typically accompanied by increases in plant biomass. Soil C dynamics, however, have shown a range of different responses to the addition of reactive N that seem to be ecosystem dependent. We evaluated the effect of N amendments on biogeochemical characteristics and microbial responses of subalpine forest organic soils in order to develop a mechanistic understanding of how soils are affected by N amendments in subalpine ecosystems. We measured a suite of responses across three years (2011–2013) during two seasons (spring and fall). Following 17 years of N amendments, fertilized soils were more acidic (control mean 5.09, fertilized mean 4.68), and had lower %C (control mean 33.7% C, fertilized mean 29.8% C) and microbial biomass C by 22% relative to control plots. Shifts in biogeochemical properties in fertilized plots were associated with an altered microbial community driven by reduced arbuscular mycorrhizal (control mean 3.2 mol%, fertilized mean 2.5 mol%) and saprotrophic fungal groups (control mean 17.0 mol%, fertilized mean 15.2 mol%), as well as a decrease in N degrading microbial enzyme activity. Our results suggest that decreases in soil C in subalpine forests were in part driven by increased microbial degradation of soil organic matter and reduced inputs to soil organic matter in the form of microbial biomass.

  1. Altering the structure and properties of iron oxide nanoparticles and graphene oxide/iron oxide composites by urea

    Energy Technology Data Exchange (ETDEWEB)

    Naghdi, Samira [Physics department, Bu-Ali Sina University, 65174 Hamedan (Iran, Islamic Republic of); Department of Mechanical Engineering, College of Engineering, Kyung Hee University, 446-701 Yongin (Korea, Republic of); Rhee, Kyong Yop, E-mail: rheeky@khu.ac.kr [Department of Mechanical Engineering, College of Engineering, Kyung Hee University, 446-701 Yongin (Korea, Republic of); Jaleh, Babak [Physics department, Bu-Ali Sina University, 65174 Hamedan (Iran, Islamic Republic of); Park, Soo Jin [Chemistry, Colloge of Natural Science, Inha University, 402-751 Incheon (Korea, Republic of)

    2016-02-28

    Graphical abstract: - Highlights: • Iron oxide (Fe{sub 2}O{sub 3}) nanoparticles were directly grown on graphene oxide (GO) using a facile microwave assistant method. • The effect of urea concentration on Fe{sub 2}O{sub 3} nanoparticles and GO/Fe{sub 2}O{sub 3} composite was examined. • Increasing urea concentration altered the morphology and decreased the particle size. • The increased concentration of urea induced a larger surface area with more active sites in the Fe{sub 2}O{sub 3} nanoparticles. • The increase in urea concentration led to decreased thermal stability of the Fe{sub 2}O{sub 3} nanoparticles. - Abstract: Iron oxide (Fe{sub 2}O{sub 3}) nanoparticles were grown on graphene oxide (GO) using a simple microwave-assisted method. The effects of urea concentration on Fe{sub 2}O{sub 3} nanoparticles and GO/Fe{sub 2}O{sub 3} composite were examined. The as-prepared samples were characterized using X-ray powder diffraction, Raman spectroscopy, and transmission electron microscopy. The Fe{sub 2}O{sub 3} nanoparticles were uniformly developed on GO sheets. The results showed that urea affects both Fe{sub 2}O{sub 3} morphology and particle size. In the absence of urea, the Fe{sub 2}O{sub 3} nanostructures exhibited a rod-like morphology. However, increasing urea concentration altered the morphology and decreased the particle size. The Raman results of GO/Fe{sub 2}O{sub 3} showed that the intensity ratio of D band to G band (I{sub D}/I{sub G}) was decreased by addition of urea, indicating that urea can preserve the GO sheets during synthesis of the composite from exposing more defects. The surface area and thermal stability of GO/Fe{sub 2}O{sub 3} and Fe{sub 2}O{sub 3} were compared using the Brunauer–Emmett–Teller method and thermal gravimetric analysis, respectively. The results showed that the increased concentration of urea induced a larger surface area with more active sites in the Fe{sub 2}O{sub 3} nanoparticles. However, the increase in urea

  2. Altering the structure and properties of iron oxide nanoparticles and graphene oxide/iron oxide composites by urea

    Science.gov (United States)

    Naghdi, Samira; Rhee, Kyong Yop; Jaleh, Babak; Park, Soo Jin

    2016-02-01

    Iron oxide (Fe2O3) nanoparticles were grown on graphene oxide (GO) using a simple microwave-assisted method. The effects of urea concentration on Fe2O3 nanoparticles and GO/Fe2O3 composite were examined. The as-prepared samples were characterized using X-ray powder diffraction, Raman spectroscopy, and transmission electron microscopy. The Fe2O3 nanoparticles were uniformly developed on GO sheets. The results showed that urea affects both Fe2O3 morphology and particle size. In the absence of urea, the Fe2O3 nanostructures exhibited a rod-like morphology. However, increasing urea concentration altered the morphology and decreased the particle size. The Raman results of GO/Fe2O3 showed that the intensity ratio of D band to G band (ID/IG) was decreased by addition of urea, indicating that urea can preserve the GO sheets during synthesis of the composite from exposing more defects. The surface area and thermal stability of GO/Fe2O3 and Fe2O3 were compared using the Brunauer-Emmett-Teller method and thermal gravimetric analysis, respectively. The results showed that the increased concentration of urea induced a larger surface area with more active sites in the Fe2O3 nanoparticles. However, the increase in urea concentration led to decreased thermal stability of the Fe2O3 nanoparticles. The magnetic properties of Fe2O3 nanoparticles were characterized by a vibrating sample magnetometer and results revealed that the magnetic properties of Fe2O3 nanoparticles are affected by the morphology.

  3. Mechano-growth factor induces migration of rat mesenchymal stem cells by altering its mechanical properties and activating ERK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jiamin; Wu, Kewen; Lin, Feng; Luo, Qing; Yang, Li; Shi, Yisong [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Song, Guanbin, E-mail: song@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Sung, Kuo-Li Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093-0412 (United States)

    2013-11-08

    Highlights: •MGF induced the migration of rat MSC in a concentration-dependent manner. •MGF enhanced the mechanical properties of rMSC in inducing its migration. •MGF activated the ERK 1/2 signaling pathway of rMSC in inducing its migration. •rMSC mechanics may synergy with ERK 1/2 pathway in MGF-induced rMSC migration. -- Abstract: Mechano-growth factor (MGF) generated by cells in response to mechanical stimulation has been identified as a mechano effector molecule, playing a key role in regulating mesenchymal stem cell (MSC) function, including proliferation and migration. However, the mechanism(s) underlying how MGF-induced MSC migration occurs is still unclear. In the present study, MGF motivated migration of rat MSCs (rMSCs) in a concentration-dependent manner and optimal concentration of MGF at 50 ng/mL (defined as MGF treatment in this paper) was demonstrated. Notably, enhancement of mechanical properties that is pertinent to cell migration, such as cell traction force and cell stiffness were found to respond to MGF treatment. Furthermore, MGF increased phosphorylation of extracellular signal-regulated kinase (ERK), ERK inhibitor (i.e., PD98059) suppressed ERK phosphorylation, and abolished MGF-induced rMSC migration were found, demonstrating that ERK is involved molecule for MGF-induced rMSC migration. These in vitro evidences of MGF-induced rMSC migration and its direct link to altering rMSC mechanics and activating the ERK pathway, uncover the underlying biomechanical and biological mechanisms of MGF-induced rMSC migration, which may help find MGF-based application of MSC in clinical therapeutics.

  4. Low concentrations of the solvent dimethyl sulphoxide alter intrinsic excitability properties of cortical and hippocampal pyramidal cells.

    Directory of Open Access Journals (Sweden)

    Francesco Tamagnini

    Full Text Available Dimethylsulfoxide (DMSO is a widely used solvent in biology. It has many applications perhaps the most common of which is in aiding the preparation of drug solutions from hydrophobic chemical entities. Recent studies have suggested that this molecule may be able to induce apoptosis in neural tissues urging caution regarding its introduction into humans, for example as part of stem cell transplants. Here we have used in vitro electrophysiological methods applied to murine brain slices to examine whether a few hours treatment with 0.05% DMSO (a concentration regarded by many as innocuous alters intrinsic excitability properties of neurones. We investigated pyramidal neurones in two distinct brain regions, namely area CA1 of the hippocampus and layer 2 of perirhinal cortex. In the former there was no effect on resting potential but input resistance was decreased by DMSO pre-treatment. In line with this action potential count for any level of depolarizing current stimulus was reduced by ∼25% following DMSO treatment. Ih-mediated "sag" was also increased in CA1 pyramids and action potential waveform analysis demonstrated that DMSO treatment moved action potential threshold towards resting potential. In perirhinal cortex a decreased action potential output for various depolarizing current stimuli was also seen. In these cells action potential threshold was unaltered by DMSO but a significant increase in action potential width was apparent. These data indicate that pre-treatment with this widely employed solvent can elicit multifaceted neurophysiological changes in mammalian neurones at concentrations below those frequently encountered in the published literature.

  5. Expression of the RNA-binding protein RBM3 is associated with a favourable prognosis and cisplatin sensitivity in epithelial ovarian cancer

    LENUS (Irish Health Repository)

    Ehlen, Asa

    2010-08-20

    Abstract Background We recently demonstrated that increased expression of the RNA-binding protein RBM3 is associated with a favourable prognosis in breast cancer. The aim of this study was to examine the prognostic value of RBM3 mRNA and protein expression in epithelial ovarian cancer (EOC) and the cisplatin response upon RBM3 depletion in a cisplatin-sensitive ovarian cancer cell line. Methods RBM3 mRNA expression was analysed in tumors from a cohort of 267 EOC cases (Cohort I) and RBM3 protein expression was analysed using immunohistochemistry (IHC) in an independent cohort of 154 prospectively collected EOC cases (Cohort II). Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between RBM3 and recurrence free survival (RFS) and overall survival (OS). Immunoblotting and IHC were used to examine the expression of RBM3 in a cisplatin-resistant ovarian cancer cell line A2780-Cp70 and its cisplatin-responsive parental cell line A2780. The impact of RBM3 on cisplatin response in EOC was assessed using siRNA-mediated silencing of RBM3 in A2780 cells followed by cell viability assay and cell cycle analysis. Results Increased RBM3 mRNA expression was associated with a prolonged RFS (HR = 0.64, 95% CI = 0.47-0.86, p = 0.003) and OS (HR = 0.64, 95% CI = 0.44-0.95, p = 0.024) in Cohort I. Multivariate analysis confirmed that RBM3 mRNA expression was an independent predictor of a prolonged RFS, (HR = 0.61, 95% CI = 0.44-0.84, p = 0.003) and OS (HR = 0.62, 95% CI = 0.41-0.95; p = 0.028) in Cohort I. In Cohort II, RBM3 protein expression was associated with a prolonged OS (HR = 0.53, 95% CI = 0.35-0.79, p = 0.002) confirmed by multivariate analysis (HR = 0.61, 95% CI = 0.40-0.92, p = 0.017). RBM3 mRNA and protein expression levels were significantly higher in the cisplatin sensitive A2780 cell line compared to the cisplatin resistant A2780-Cp70 derivative. siRNA-mediated silencing of RBM3 expression in the A2780 cells resulted

  6. A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis

    KAUST Repository

    Chen, Tao

    2013-10-17

    Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges. © 2013 Chen et al.

  7. Alterations in composition of sterols and in properties of erythrocyte membranes in rats with Shvetz experimental leukosis and after UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Palamarchyuk, V.I.; Trikash, I.O. (AN Ukrainskoj SSR, Kiev. Inst. Biokhimii)

    1983-05-01

    Sterol composition of erythrocyte membrane is studied in experimental Schwetz leukosis. Interconnections of alteration in sterol composition and membrane properties are investigated, as well as the effect of UV-radiation on the alteration of sterol composition of erythrocyte membrane and lifetime of animals With leukosis. The effect of UV-radiation on survival of the leukosis animals has been studied which showed that irradiation of rats in the dose of 8 mWt/min/cm/sup 2/ for 12 days increases the lifetime of animals by several days, i.e. increases resistance of animals to leukosis. In the case of short-time UV-radiation of rats the cholesterine amount in erythrocyte membranes increases by 9% as compared with the norm, simultaneously, other substances appear. Acidic resistance of erythrocytes of irradiated animals also increases. The supposition is made that alterations promote the increase of organism resistance to leukosis.

  8. Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export* S⃞

    OpenAIRE

    Fasken, Milo B.; Stewart, Murray; Corbett, Anita H.

    2008-01-01

    Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal...

  9. TIAR and TIA-1 mRNA binding proteins co-aggregate under conditions of rapid oxygen decline and extreme hypoxia, suppress HIF-1alpha pathway and inhibit proliferation and angiogenesis

    OpenAIRE

    Gottschald, Oana Raluca

    2010-01-01

    T-cell intracellular antigen (TIA)-1 and TIA-1 related protein (TIAR) are mRNA-binding proteins that aggregate within stress granules under specific stress conditions. In this study, we analyzed TIAR/TIA-1 aggregation under different hypoxic conditions, and studied the effects on hypoxia-inducible factor (HIF)-1alpha, as well as on proliferation and angiogenesis. TIAR/TIA-1 formed stress granules under acute and pronounced hypoxic conditions in A549 adenocarcinoma cells. In parallel, HIF-1alp...

  10. Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

    Institute of Scientific and Technical Information of China (English)

    LIANG; Xingjie; (

    2001-01-01

    , 1146(1): 136.[12] Howlett, N. G., Avery, S. V., Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsatu-ration in Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol., 1997, 48(4): 539.[13] Petriz, J., Oconnor, J. E., Carmona, M. et al., Is Rhodamine-123 an appropriate fluorescent probe to assess P-glycoprotein mediated multidrug resistance in vinblastine-resistant CHO cells? Analytical Cellular Pathology, 1997, 14(3): 129.[14] Leonce, S., Burbridge, M., Flow cytometry: a useful technique in the study of multidrug resistance, J. Bio. Cell, 1993, 78(1-2): 63.[15] Le Moyec, L., Tatoud, R., Degorges, A. et al., Proton nuclear magnetic resonance spectroscopy reveals cellular lipids in-volved in resistance to Adriamycin and Taxol by the K562 Leukemia cell line, Cancer Res., 1996, 56: 3461.[16] Callaghan, R., Stafford, A., Epand, R. M., Increased accumulation of drugs in a multidrug resistant cell line by alteration of membrane biophysical properties, Biochim. Biophys. Acta, 1993, 1175(3): 277.[17] Sinicrope, F. A., Dudeia, P. K., Bissommette, B. M. et al., Modulation of P-glycoprotein-mediated drug transport by al-terations in lipid fluidity of rat liver canlicular membrane vesicles, J. Biol. Chem., 1992, 267(35): 24995.[18] Romsicki, Y., Sharom, F. J., The membrane lipid environment modulates drug interactions with the P-glycoprotein multi-drug transporter, Biochemistry, 1999, 38(21): 6887.[19] Garel, O., Lecureur, V., Guillouzo, A., The P-glycoprotein multidrug transporter, Gen. Pharmacol., 1996, 27(8): 1283.[20] Aran, J. M., Pastan, I., Gottesman, M. M., Therapeutic strategies involving the multidrug resistance phenotype: the MDR1 gene as target, chemoprotectant, and selectable marker in gene therapy, Adv. Pharmacol., 1999, 46: 1.[21] Zaman, G. J., Flens, M. J., Vanleusden, M. R. et al., The human multidrug resistance-associated protein (MRP) is a plasma membrane drug efflux pump, Proc. Natl. Acad

  11. Insights into magmatic processes and hydrothermal alteration of in situ superfast spreading ocean crust at ODP/IODP site 1256 from a cluster analysis of rock magnetic properties

    Science.gov (United States)

    Dekkers, Mark J.; Heslop, David; Herrero-Bervera, Emilio; Acton, Gary; Krasa, David

    2014-08-01

    analyze magnetic properties from Ocean Drilling Program (ODP)/Integrated ODP (IODP) Hole 1256D (6°44.1' N, 91°56.1' W) on the Cocos Plate in ˜15.2 Ma oceanic crust generated by superfast seafloor spreading, the only drill hole that has sampled all three oceanic crust layers in a tectonically undisturbed setting. Fuzzy c-means cluster analysis and nonlinear mapping are utilized to study down-hole trends in the ratio of the saturation remanent magnetization and the saturation magnetization, the coercive force, the ratio of the remanent coercive force and coercive force, the low-field magnetic susceptibility, and the Curie temperature, to evaluate the effects of magmatic and hydrothermal processes on magnetic properties. A statistically robust five cluster solution separates the data predominantly into three clusters that express increasing hydrothermal alteration of the lavas, which differ from two distinct clusters mainly representing the dikes and gabbros. Extensive alteration can obliterate magnetic property differences between lavas, dikes, and gabbros. The imprint of thermochemical alteration on the iron-titanium oxides is only partially related to the porosity of the rocks. Thus, the analysis complements interpretation based on electrofacies analysis. All clusters display rock magnetic characteristics compatible with an ability to retain a stable natural remanent magnetization suggesting that the entire sampled sequence of ocean crust can contribute to marine magnetic anomalies. Paleointensity determination is difficult because of the propensity of oxyexsolution during laboratory heating and/or the presence of intergrowths. The upper part of the extrusive sequence, the granoblastic dikes, and moderately altered gabbros may contain a comparatively uncontaminated thermoremanent magnetization.

  12. Altered age-related changes in bioenergetic properties and mitochondrial morphology in fibroblasts from sporadic amyotrophic lateral sclerosis patients.

    Science.gov (United States)

    Allen, Scott P; Duffy, Lynn M; Shaw, Pamela J; Grierson, Andrew J

    2015-10-01

    Mitochondria play a key role in aging, which is a well-established risk factor in amyotrophic lateral sclerosis (ALS). We have previously modeled metabolic dysregulation in ALS using fibroblasts isolated from sporadic ALS (SALS) and familial ALS patients. In the present study, we show that fibroblasts from SALS patients have an altered metabolic response to aging. Control fibroblasts demonstrated increased mitochondrial network complexity and spare respiratory capacity with age which was not seen in the SALS cases. SALS cases displayed an increase in uncoupled mitochondrial respiration, which was not evident in control cases. Unlike SALS cases, controls showed a decrease in glycolysis and an increase in the oxygen consumption rate/extracellular acidification rate ratio, indicating an increased reliance on mitochondrial function. Switching to a more oxidative state by removing glucose with in the culture media resulted in a loss of the mitochondrial interconnectivity and spare respiratory capacity increases observed in controls grown in glucose. Glucose removal also led to an age-independent increase in glycolysis in the SALS cases. This study is, to the best our knowledge, the first to assess the effect of aging on both mitochondrial and glycolytic function simultaneously in intact human fibroblasts and demonstrates that the SALS disease state shifts the cellular metabolic response to aging to a more glycolytic state compared with age-matched control fibroblasts. This work highlights that ALS alters the metabolic equilibrium even in peripheral tissues outside the central nervous system. Elucidating at a molecular level how this occurs and at what stage in the disease process is crucial to understanding why ALS affects cellular energy metabolism and how the disease alters the natural cellular response to aging.

  13. [Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex].

    Science.gov (United States)

    Babkina, G T; Karpova, G G; Matasova, N B; Berzin', V M; Gren, E Ia

    1985-01-01

    2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.

  14. Alteration of Mesoscopic Properties and Mechanical Behavior of Sandstone Due to Hydro-Physical and Hydro-Chemical Effects

    Science.gov (United States)

    Qiao, Liping; Wang, Zhechao; Huang, Anda

    2017-02-01

    The hydro-physical and hydro-chemical interactions between groundwater and a rock mass can lead to changes in the mineral composition and structure of the rock (e.g., generation of voids and dissolution pores and an increase in the porosity), thereby altering the macroscopic mechanical characteristics of the rock mass. Sandstone specimens were saturated with distilled water and five aqueous solutions characterized by various ion concentrations and pH values for several months, and their porosity was measured in real time. Simultaneously, the concentration and pH of each aqueous solution were monitored every 30 days. The results indicate that after immersion in the aqueous solutions for 180 days, the porosity of the sandstone specimens and the ion concentrations and pH of the aqueous solutions tended to stabilize. Then, the immersed sandstone specimens were analyzed in thin section and subjected to computerized tomography scanning. It turns out that the mineral composition and structure of the specimens had all changed to various degrees. Finally, the uniaxial compression tests were conducted on the sandstone specimens to analyze the effects of the hydro-physical and hydro-chemical alteration on the macroscopic mechanical characteristics of the rock (e.g., the stress-strain relationship, elastic modulus, and peak strength). The results of this study can serve as a reference for investigations into theories and applications of water-rock interactions and for research in related fields.

  15. Density, porosity, mineralogy, and internal structure of cosmic dust and alteration of its properties during high velocity atmospheric entry

    CERN Document Server

    Kohout, T; Suuronen, J -P; Rochette, P; Hutzler, A; Gattacceca, J; Skála, D D Badjukov R; Böhmová, V; Čuda, J

    2014-01-01

    X-ray microtomography (XMT), X-ray diffraction (XRD) and magnetic hysteresis measurements were used to determine micrometeorite internal structure, mineralogy, crystallography, and physical properties at ~{\\mu}m resolution. The study samples include unmelted, partially melted (scoriaceous) and completely melted (cosmic spherules) micrometeorites. This variety not only allows comparison of the mineralogy and porosity of these three micrometeorite types, but also reveals changes in meteoroid properties during atmospheric entry at various velocities. At low entry velocities, meteoroids do not melt, and their physical properties do not change. The porosity of unmelted micrometeorites varies considerably (0-12%) with one friable example having porosity around 50%. At higher velocities, the range of meteoroid porosity narrows, but average porosity increases (to 16-27%) due to volatile evaporation and partial melting (scoriaceous phase). Metal distribution seems to be mostly unaffected at this stage. At even higher ...

  16. Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Hynes, M.D.; Lochner, M.A.; Bemis, K.G.; Hymson, D.L.

    1985-06-17

    The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with /sup 3/H-naloxone or /sup 3/H-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties. 30 references, 2 figures, 2 tables.

  17. Sulfation of a polysaccharide produced by a marine filamentous fungus Phoma herbarum YS4108 alters its antioxidant properties in vitro.

    Science.gov (United States)

    Yang, X B; Gao, X D; Han, F; Tan, R X

    2005-08-30

    Free radicals and other reactive oxygen species (ROS) are generated by all aerobic cells and are widely believed to play a significant role in aging as well as a number of degenerative or pathological diseases. This study compared the free radical-scavenging properties and antioxidant activity of YCP, a polysaccharide from the mycelium of a marine filamentous fungus Phoma herbarum YS 4108 and its two chemically sulfated derivatives YCP-S1 and YCP-S2. Sulfation, which masks hydroxyl groups of YCP polysaccharide molecule, could introduce new antioxidant activity, such as superoxide and hydroxyl radicals scavenging activity, metal chelating action, lipid peroxidation and linoleic acid oxidation inhibition capability. Furthermore, sulfated YCP was more potent than YCP at protecting erythrocytes against oxidative damage hemolysis. The current data suggest for the first time that sulfation of polysaccharide significantly increases its antioxidant activity and the chemical modification of polysaccharides may allow the preparation of derivatives with new properties and a variety of applications.

  18. Exercise Training after Spinal Cord Injury Selectively Alters Synaptic Properties in Neurons in Adult Mouse Spinal Cord

    Science.gov (United States)

    Flynn, Jamie R.; Dunn, Lynda R.; Galea, Mary P.; Callister, Robin; Rank, Michelle M.

    2013-01-01

    Abstract Following spinal cord injury (SCI), anatomical changes such as axonal sprouting occur within weeks in the vicinity of the injury. Exercise training enhances axon sprouting; however, the exact mechanisms that mediate exercised-induced plasticity are unknown. We studied the effects of exercise training after SCI on the intrinsic and synaptic properties of spinal neurons in the immediate vicinity (<2 segments) of the SCI. Male mice (C57BL/6, 9–10 weeks old) received a spinal hemisection (T10) and after 1 week of recovery, they were randomized to trained (treadmill exercise for 3 weeks) and untrained (no exercise) groups. After 3 weeks, mice were killed and horizontal spinal cord slices (T6–L1, 250 μm thick) were prepared for visually guided whole cell patch clamp recording. Intrinsic properties, including resting membrane potential, input resistance, rheobase current, action potential (AP) threshold and after-hyperpolarization (AHP) amplitude were similar in neurons from trained and untrained mice (n=67 and 70 neurons, respectively). Neurons could be grouped into four categories based on their AP discharge during depolarizing current injection; the proportions of tonic firing, initial bursting, single spiking, and delayed firing neurons were similar in trained and untrained mice. The properties of spontaneous excitatory synaptic currents (sEPSCs) did not differ in trained and untrained animals. In contrast, evoked excitatory synaptic currents recorded after dorsal column stimulation were markedly increased in trained animals (peak amplitude 78.9±17.5 vs. 42.2±6.8 pA; charge 1054±376 vs. 348±75 pA·ms). These data suggest that 3 weeks of treadmill exercise does not affect the intrinsic properties of spinal neurons after SCI; however, excitatory synaptic drive from dorsal column pathways, such as the corticospinal tract, is enhanced. PMID:23320512

  19. Morphological alteration and exceptional magnetic properties of air-stable FeCo nanocubes prepared by a chemical reduction method

    Energy Technology Data Exchange (ETDEWEB)

    Chokprasombat, K., E-mail: komkrich28@gmail.com [School of Physics, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand); Harding, P. [Molecular Technology Research Unit, School of Science, Walailak University, Nakhon Si Thammarat 80161 (Thailand); Pinitsoontorn, S. [Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002 (Thailand); Maensiri, S. [School of Physics, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand)

    2014-11-15

    FeCo nanocubes are of great interest due to their outstanding magnetic properties and larger contact area compared to the spherical particles. Herein, the FeCo nanocubes could be obtained by the reduction of metal ions by hydrazine hydrate under a concentrated basic condition. It was found that shape of the FeCo nanocubes varied from cubic with a mean edge length of 130±3 nm to polyhedron (diameter around 500–700 nm) depending on the concentration of using polymer. A lot of irregular nanoplates were also obtained when used the excessive polymer. In addition, the as-synthesized particles were air-stable which might be related to the formation of thin polymer shells on particle surfaces. The FeCo nanocubes also possessed exceptional magnetic properties at room temperature, including a very high saturation magnetization (217.14 emu/g) and low coercivity (85.95 Oe). - Highlights: • We report the synthesis of FeCo nanocubes by a chemical reduction method. • Shape of the particles clearly depended on the amount of polymer used. • The particles were air-stable and possessed excellent magnetic properties.

  20. The adrenal specific toxicant mitotane directly interacts with lipid membranes and alters membrane properties depending on lipid composition.

    Science.gov (United States)

    Scheidt, Holger A; Haralampiev, Ivan; Theisgen, Stephan; Schirbel, Andreas; Sbiera, Silviu; Huster, Daniel; Kroiss, Matthias; Müller, Peter

    2016-06-15

    Mitotane (o,p'.-DDD) is an orphan drug approved for the treatment of adrenocortical carcinoma. The mechanisms, which are responsible for this activity of the drug, are not completely understood. It can be hypothesized that an impact of mitotane is mediated by the interaction with cellular membranes. However, an interaction of mitotane with (lipid) membranes has not yet been investigated in detail. Here, we characterized the interaction of mitotane and its main metabolite o,p'-dichlorodiphenyldichloroacetic acid (o,p'-DDA) with lipid membranes by applying a variety of biophysical approaches of nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that mitotane and o,p'-DDA bind to lipid membranes by inserting into the lipid-water interface of the bilayer. Mitotane but not o,p'-DDA directly causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. Mitotane induced alterations of the membrane integrity required the presence of phosphatidylethanolamine and/or cholesterol. Collectively, our data for the first time characterize the impact of mitotane on the lipid membrane structure and dynamics, which may contribute to a better understanding of specific mitotane effects and side effects.

  1. Prenatal drug exposure to illicit drugs alters working memory-related brain activity and underlying network properties in adolescence.

    Science.gov (United States)

    Schweitzer, Julie B; Riggins, Tracy; Liang, Xia; Gallen, Courtney; Kurup, Pradeep K; Ross, Thomas J; Black, Maureen M; Nair, Prasanna; Salmeron, Betty Jo

    2015-01-01

    The persistence of effects of prenatal drug exposure (PDE) on brain functioning during adolescence is poorly understood. We explored neural activation to a visuospatial working memory (VSWM) versus a control task using functional magnetic resonance imaging (fMRI) in adolescents with PDE and a community comparison group (CC) of non-exposed adolescents. We applied graph theory metrics to resting state data using a network of nodes derived from the VSWM task activation map to further explore connectivity underlying WM functioning. Participants (ages 12-15 years) included 47 adolescents (27 PDE and 20 CC). All analyses controlled for potentially confounding differences in birth characteristics and postnatal environment. Significant group by task differences in brain activation emerged in the left middle frontal gyrus (BA 6) with the CC group, but not the PDE group, activating this region during VSWM. The PDE group deactivated the culmen, whereas the CC group activated it during the VSWM task. The CC group demonstrated a significant relation between reaction time and culmen activation, not present in the PDE group. The network analysis underlying VSWM performance showed that PDE group had lower global efficiency than the CC group and a trend level reduction in local efficiency. The network node corresponding to the BA 6 group by task interaction showed reduced nodal efficiency and fewer direct connections to other nodes in the network. These results suggest that adolescence reveals altered neural functioning related to response planning that may reflect less efficient network functioning in youth with PDE.

  2. Age- and gender-related distribution of bone mineral density and mechanical properties of the proximal humerus; Alters- und geschlechtsabhaengige Knochenmineraldichteverteilung und mechanische Eigenschaften des proximalen Humerus

    Energy Technology Data Exchange (ETDEWEB)

    Lill, H.; Hepp, P.; Korner, J.; Josten, C. [Klinik fuer Unfall- und Wiederherstellungschirurgie, Univ. Leipzig (Germany); Gowin, W. [Center of Muscle and Bone Research, Klinik fuer Radiologie und Nuklearmedizin, Universitaetsklinikum Benjamin Franklin, Freie Univ. Berlin (Germany); Oestmann, J.W. [Klinik fuer Radiologie, Charite, Virchow-Klinikum, Humboldt Univ., Berlin (Germany); Haas, N.P.; Duda, G.N. [Klinik fuer Unfall- und Wiederherstellungschirurgie, Charite, Virchow-Klinikum Humboldt-Univ. Berlin (Germany)

    2002-12-01

    Purpose: To evaluate age- and gender-related mechanical properties and bone mineral density (BMD) of the proximal humerus at different levels and regions. Materials and methods: Mechanical indentation testing, DXA, QCT, pQCT and the radiogrammetry (Cortical Index, CI) were carried out in 70 freshly harvested humeri from 46 human cadavers (23 females, 23-males; median age 70.5 years). Results: In the female group, a high correlation between age and BMD was found ({rho}=0.62 to -0.70, p<0.01) with statistically significant differences between specimens of patients 69 years or younger, and 70 years or older (p<0.05). In the group of female specimens of age 70 years or older, BMD values were found to be significantly lower compared to their male counterparts (p<0.05). Regardless of the specimen's age, the highest BMD and bone strength were found in the proximal aspect and in the medial and dorsal regions of the proximal humerus. Conclusion: These findings provide an insight into the fracture mechanism of the proximal humerus and should be the basis for designing structure-oriented implants with improved implant-bone stability in osteoporotic patients. (orig.) [German] Ziel: Das Ziel der vorliegenden Studie war die alters- und geschlechtsspezifische Analyse der mechanischen Eigenschaften und der Knochenmineraldichte (BMD) des proximalen Humerus in verschiedenen Hoehen und Regionen. Methoden: Folgende Verfahren wurden angewandt: Mechanische Indentation Testung, DXA, QCT, pQCT und die Radiogrammetrie (Cortical Index, CI). Die Untersuchungen wurden an 70 frischen Humeri von 46 humanen Praeparaten (23 weiblich, 23 maennlich; Alter median: 70,5 Jahre) durchgefuehrt. Ergebnisse: In der Gruppe der weiblichen Humeri fand sich eine hohe Korrelation zwischen Alter und Knochenmineraldichte ({rho}=-0,62 to -0,70 p<0,01) mit statistisch signifikanten Unterschieden zwischen Praeparaten juenger als 69 Jahre und aelter als 70 Jahre (p<0.05). In der Gruppe der weiblichen Praeparate

  3. Reduced Neck Muscle Strength and Altered Muscle Mechanical Properties in Cervical Dystonia Following Botulinum Neurotoxin Injections: A Prospective Study

    Science.gov (United States)

    Mustalampi, Sirpa; Ylinen, Jari; Korniloff, Katariina; Weir, Adam; Häkkinen, Arja

    2016-01-01

    Objective To evaluate changes in the strength and mechanical properties of neck muscles and disability in patients with cervical dystonia (CD) during a 12-week period following botulinum neurotoxin (BoNT) injections. Methods Eight patients with CD volunteered for this prospective clinical cohort study. Patients had received BoNT injections regularly in neck muscles at three-month intervals for several years. Maximal isometric neck strength was measured by a dynamometer, and the mechanical properties of the splenius capitis were evaluated using two myotonometers. Clinical assessment was performed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) before and at 2, 4, 8, and 12 weeks after the BoNT injections. Results Mean maximal isometric neck strength at two weeks after the BoNT injections decreased by 28% in extension, 25% in rotation of the affected side and 17% in flexion. At four weeks, muscle stiffness of the affected side decreased by 17% and tension decreased by 6%. At eight weeks, the muscle elasticity on the affected side increased by 12%. At two weeks after the BoNT injections, the TWSTRS-severity and TWSTRS-total scores decreased by 4.3 and 6.4, respectively. The strength, muscle mechanical properties and TWSTRS scores returned to baseline values at 12 weeks. Conclusions Although maximal neck strength and muscle tone decreased after BoNT injections, the disability improved. The changes observed after BoNT injections were temporary and returned to pre-injection levels within twelve weeks. Despite having a possible negative effect on function and decreasing neck strength, the BoNT injections improved the patients reported disability. PMID:26828215

  4. Reduced Neck Muscle Strength and Altered Muscle Mechanical Properties in Cervical Dystonia Following Botulinum Neurotoxin Injections: A Prospective Study

    Directory of Open Access Journals (Sweden)

    Sirpa Mustalampi

    2016-01-01

    Full Text Available Objective To evaluate changes in the strength and mechanical properties of neck muscles and disability in patients with cervical dystonia (CD during a 12-week period following botulinum neurotoxin (BoNT injections. Methods Eight patients with CD volunteered for this prospective clinical cohort study. Patients had received BoNT injections regularly in neck muscles at three-month intervals for several years. Maximal isometric neck strength was measured by a dynamometer, and the mechanical properties of the splenius capitis were evaluated using two myotonometers. Clinical assessment was performed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS before and at 2, 4, 8, and 12 weeks after the BoNT injections. Results Mean maximal isometric neck strength at two weeks after the BoNT injections decreased by 28% in extension, 25% in rotation of the affected side and 17% in flexion. At four weeks, muscle stiffness of the affected side decreased by 17% and tension decreased by 6%. At eight weeks, the muscle elasticity on the affected side increased by 12%. At two weeks after the BoNT injections, the TWSTRS-severity and TWSTRS-total scores decreased by 4.3 and 6.4, respectively. The strength, muscle mechanical properties and TWSTRS scores returned to baseline values at 12 weeks. Conclusions Although maximal neck strength and muscle tone decreased after BoNT injections, the disability improved. The changes observed after BoNT injections were temporary and returned to pre-injection levels within twelve weeks. Despite having a possible negative effect on function and decreasing neck strength, the BoNT injections improved the patients reported disability.

  5. Behavioral and molecular neuroepigenetic alterations in prenatally stressed mice: relevance for the study of chromatin remodeling properties of antipsychotic drugs

    Science.gov (United States)

    Dong, E; Tueting, P; Matrisciano, F; Grayson, D R; Guidotti, A

    2016-01-01

    We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg−1 twice a day for 5 days) but not by haloperidol (1 mg kg−1 twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention. PMID:26756904

  6. Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II.

    Science.gov (United States)

    Umate, Pavan; Schwenkert, Serena; Karbat, Izhar; Dal Bosco, Cristina; Mlcòchová, Lada; Volz, Stefanie; Zer, Hagit; Herrmann, Reinhold G; Ohad, Itzhak; Meurer, Jörg

    2007-03-30

    Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

  7. Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

    Science.gov (United States)

    Nakamura, Yukiko; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2003-01-01

    The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice (Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10-50 degrees C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30-40 degrees C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40 degrees C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30-40 degrees C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40 degrees C were substantially higher than those grown at 10, 20 and 50 degrees C. Furthermore, the activities of (1-->3),(1-->4)-beta-glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1-->3),(1-->4)-beta-glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30-40 degrees C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of beta -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of beta-glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.

  8. Alterations in energy properties of eucalyptus wood and bark subjected to torrefaction: the potential of mass loss as a synthetic indicator.

    Science.gov (United States)

    Almeida, G; Brito, J O; Perré, P

    2010-12-01

    Torrefaction is a mild pyrolysis process (usually up to 300 degrees C) that changes the chemical and physical properties of biomass. This process is a possible pre-treatment prior to further processes (transport, grinding, combustion, gasification, etc) to generate energy or biofuels. In this study, three eucalyptus wood species and bark were subjected to different torrefaction conditions to determine the alterations in their structural and energy properties. The most severe treatment (280 degrees C, 5h) causes mass losses of more than 35%, with severe damage to anatomical structure, and an increase of about 27% in the specific energy content. Bark is more sensitive to heat than wood. Energy yields are always higher than mass yields, thereby demonstrating the benefits of torrefaction in concentrating biomass energy. The overall mass loss is proposed as a relevant parameter to synthesize the effect of torrefaction conditions (temperature and duration). Accordingly, all results are summarised by analytical expressions able to predict the energy properties as a function of the overall mass loss. These expressions are intended to be used in any optimization procedure, from production in the field to the final use.

  9. A high mixed protein diet reduces body fat without altering the mechanical properties of bone in female rats.

    Science.gov (United States)

    Pye, Kathleen M; Wakefield, Andrew P; Aukema, Harold M; House, James D; Ogborn, Malcolm R; Weiler, Hope A

    2009-11-01

    Long-term consumption of high-protein (HP) diets at 35% of energy is postulated to negatively influence bone health. Previous studies have not comprehensively examined the biochemical, physical, and biomechanical properties of bone required to arrive at this conclusion. Our objective in this study was to examine the long-term effect of a HP diet on bone metabolism, mass, and strength in rats. Adult female Sprague-Dawley rats (n = 80) were randomized to receive for 4, 8, 12, or 17 mo a normal-protein (NP) control diet (15% of energy) or a HP diet (35% of energy). Diets were balanced for calcium because the protein sources were rich in calcium. At each time point, measurements included weight, body composition, and bone mass using dual-energy X-ray absorptiometry, mechanical strength at the mid-diaphysis of femur and tibia, microarchitecture of femurs using microcomputerized tomography and serum osteocalcin, carboxy-terminal crosslinks of type I collagen (CTX), insulin-like growth factor-1 (IGF-1), leptin, and adiponectin. Effects of diet, time, and their interaction were tested using factorial ANOVA. The HP diet resulted in lower body weight, total body, and abdominal fat and higher lean mass. Serum leptin and adiponectin were greater in HP-fed than in NP-fed rats, but IGF-1 did not differ between the groups. Whereas the HP diet resulted in higher relative bone mineral content (g/kg) in the femur, tibia, and vertebrae, serum osteocalcin and CTX and bone internal architecture and biomechanical strength were unaffected. In conclusion, HP diets at 35% of energy lower body fat content without hindering the mechanical and weight-bearing properties of bone.

  10. E series prostaglandins alter the proliferative, apoptotic and migratory properties of T98G human glioma cells in vitro

    Directory of Open Access Journals (Sweden)

    Gomes Renata N

    2012-12-01

    Full Text Available Abstract Background In many types of cancer, prostaglandin E2 (PGE2 is associated with tumour related processes including proliferation, migration, angiogenesis and apoptosis. However in gliomas the role of this prostanoid is poorly understood. Here, we report on the proliferative, migratory, and apoptotic effects of PGE1, PGE2 and Ibuprofen (IBP observed in the T98G human glioma cell line in vitro. Methods T98G human glioma cells were treated with IBP, PGE1 or PGE2 at varying concentrations for 24–72 hours. Cell proliferation, mitotic index and apoptotic index were determined for each treatment. Caspase-9 and caspase-3 activity was measured using fluorescent probes in live cells (FITC-LEHD-FMK and FITC-DEVD-FMK respectively. The migratory capacity of the cells was quantified using a scratch migration assay and a transwell migration assay. Results A significant decrease was seen in cell number (54% in the presence of 50 μM IBP. Mitotic index and bromodeoxyuridine (BrdU incorporation were also decreased 57% and 65%, respectively, by IBP. The apoptotic index was increased (167% and the in situ activity of caspase-9 and caspase-3 was evident in IBP treated cells. The inhibition of COX activity by IBP also caused a significant inhibition of cell migration in the monolayer scratch assay (74% and the transwell migration assay (36%. In contrast, the presence of exogenous PGE1 or PGE2 caused significant increases in cell number (37% PGE1 and 45% PGE2. When mitotic index was measured no change was found for either PG treatment. However, the BrdU incorporation rate was significantly increased by PGE1 (62% and to a greater extent by PGE2 (100%. The apoptotic index was unchanged by exogenous PGs. The addition of exogenous PGs caused an increase in cell migration in the monolayer scratch assay (43% PGE1 and 44% PGE2 and the transwell migration assay (28% PGE1 and 68% PGE2. Conclusions The present study demonstrated that treatments which alter PGE1 and PGE

  11. Evaluation of surface properties and atmospheric disturbances caused by post-dam alterations of land use/land cover

    Science.gov (United States)

    Woldemichael, A. T.; Hossain, F.; Pielke, R., Sr.

    2014-09-01

    This study adopted a differential land-use/land-cover (LULC) analysis to evaluate dam-triggered land-atmosphere interactions for a number of LULC scenarios. Two specific questions were addressed: (1) can dam-triggered LULC heterogeneities modify surface and energy budget, which, in turn, change regional convergence and precipitation patterns? (2) How extensive is the modification in surface moisture and energy budget altered by dam-triggered LULC changes occurring in different climate and terrain features? The Regional Atmospheric Modeling System (RAMS, version 6.0) was set up for two climatologically and topographically contrasting regions: the American River watershed (ARW), located in California, and the Owyhee River watershed (ORW), located in eastern Oregon. For the selected atmospheric river precipitation event of 29 December 1996 to 3 January 1997, simulations of three pre-defined LULC scenarios are performed. The definition of the scenarios are (1) the "control" scenario, representing the contemporary land use, (2) the "pre-dam" scenario, representing the natural landscape before the construction of the dams and (3) the "non-irrigation" scenario, representing the condition where previously irrigated landscape in the control is transformed to the nearby land-use type. Results indicated that the ARW energy and moisture fluxes were more extensively affected by dam-induced changes in LULC than the ORW. Both regions, however, displayed commonalities in the modification of land-atmosphere processes due to LULC changes, with the control-non-irrigation scenario creating more change than the control-pre-dam scenarios. These commonalities were: (1) the combination of a decrease in temperature (up to 0.15 °C) and an increase at dew point (up to 0.25 °C) was observed; (2) there was a larger fraction of energy partitioned to latent heat flux (up to 10 W m-2) that increased the amount of water vapor in the atmosphere and resulted in a larger convective available

  12. Evaluation of surface properties and atmospheric disturbances caused by post-dam alterations of land-use/land-cover

    Science.gov (United States)

    Woldemichael, A. T.; Hossain, F.; Pielke, R., Sr.

    2014-05-01

    This study adopted a differential land-use/land-cover (LULC) analysis to evaluate dam-triggered land-atmosphere interactions for a number of LULC scenarios. Two specific questions were addressed: (1) can dam-triggered LULC heterogeneities modify surface and energy budget which, in turn, change regional convergence and precipitation patterns? and (2) how extensive is the modification in surface moisture and energy budget altered by dam-triggered LULC changes occurring in different climate and terrain features? The Regional Atmospheric Modeling System (RAMS, version 6.0) was set up for two climatologically and topographically contrasting regions: the American River Watershed (ARW) located in California and the Owyhee River Watershed (ORW) located in eastern Oregon. For the selected atmospheric river precipitation event of 29 December 1996 to 3 January 1997, simulations of three pre-defined LULC scenarios are performed. The definition of the scenarios are: (1) the control scenario representing the contemporary land-use, (2) the pre-dam scenario representing the natural landscape before the construction of the dams and (3) the non-irrigation scenario representing the condition where previously irrigated landscape in the control is transformed to the nearby land-use type. Results indicated that the ARW energy and moisture fluxes were more extensively affected by dam-induced changes in LULC than the ORW. Both regions, however, displayed commonalities in the modification of land-atmosphere processes due to LULC changes, with the control-non-irrigation scenario creating more change than the control-pre-dam scenarios. These commonalities were: (1) the combination of a decrease in temperature (up to 0.15 °C) and an increase in dewpoint (up to 0.25 °C) was observed, (2) there was a larger fraction of energy partitioned to latent heat flux (up to 10 W m-2) that increased the amount of water vapor to the atmosphere and resulted in a larger convective available potential

  13. Evaluation of surface properties and atmospheric disturbances caused by post-dam alterations of land-use/land-cover

    Directory of Open Access Journals (Sweden)

    A. T. Woldemichael

    2014-05-01

    Full Text Available This study adopted a differential land-use/land-cover (LULC analysis to evaluate dam-triggered land–atmosphere interactions for a number of LULC scenarios. Two specific questions were addressed: (1 can dam-triggered LULC heterogeneities modify surface and energy budget which, in turn, change regional convergence and precipitation patterns? and (2 how extensive is the modification in surface moisture and energy budget altered by dam-triggered LULC changes occurring in different climate and terrain features? The Regional Atmospheric Modeling System (RAMS, version 6.0 was set up for two climatologically and topographically contrasting regions: the American River Watershed (ARW located in California and the Owyhee River Watershed (ORW located in eastern Oregon. For the selected atmospheric river precipitation event of 29 December 1996 to 3 January 1997, simulations of three pre-defined LULC scenarios are performed. The definition of the scenarios are: (1 the control scenario representing the contemporary land-use, (2 the pre-dam scenario representing the natural landscape before the construction of the dams and (3 the non-irrigation scenario representing the condition where previously irrigated landscape in the control is transformed to the nearby land-use type. Results indicated that the ARW energy and moisture fluxes were more extensively affected by dam-induced changes in LULC than the ORW. Both regions, however, displayed commonalities in the modification of land–atmosphere processes due to LULC changes, with the control–non-irrigation scenario creating more change than the control–pre-dam scenarios. These commonalities were: (1 the combination of a decrease in temperature (up to 0.15 °C and an increase in dewpoint (up to 0.25 °C was observed, (2 there was a larger fraction of energy partitioned to latent heat flux (up to 10 W m−2 that increased the amount of water vapor to the atmosphere and resulted in a larger convective available

  14. Genetic deletion of TREK-1 or TWIK-1/TREK-1 potassium channels does not alter the basic electrophysiological properties of mature hippocampal astrocytes in situ

    Directory of Open Access Journals (Sweden)

    Yixing eDu

    2016-02-01

    Full Text Available We have recently shown that a linear current-to-voltage (I-V relationship of membrane conductance (passive conductance reflects the intrinsic property of K+ channels in mature astrocytes. While passive conductance is known to underpin a highly negative and stable membrane potential (VM essential for the basic homeostatic function of astrocytes, a complete repertoire of the involved K+ channels remains elusive. TREK-1 two-pore domain K+ channel (K2P is highly expressed in astrocytes, and covalent association of TREK-1 with TWIK-1, another highly expressed astrocytic K2P, has been reported as a mechanism underlying the trafficking of this heterodimer channel to the membrane and contributing to astrocytes’ passive conductance. To decipher the individual contribution of TREK-1 and address whether the appearance of passive conductance is conditional to the co-expression of TWIK-1/TREK-1 in astrocytes, TREK-1 single and TWIK-1/TREK-1 double gene knockout mice were used in the present study. The relative quantity of mRNA encoding other astrocyte K+ channels, such as Kir4.1, Kir5.1, and TREK-2, was not altered in these gene knockout mice. Whole-cell recording from hippocampal astrocytes in situ revealed no detectable changes in astrocyte passive conductance, VM, or membrane input resistance (Rin in either kind of gene knockout mouse. Additionally, TREK-1 proteins were mainly located in the intracellular compartments of the hippocampus. Altogether, genetic deletion of TREK-1 alone or together with TWIK-1 produced no obvious alteration in the basic electrophysiological properties of hippocampal astrocytes. Thus, future research focusing on other K+ channels may shed light on this long-standing and important question in astrocyte physiology.

  15. Housing conditions alter properties of the tibia and humerus during the laying phase in Lohmann white Leghorn hens.

    Science.gov (United States)

    Regmi, P; Smith, N; Nelson, N; Haut, R C; Orth, M W; Karcher, D M

    2016-01-01

    Osteoporosis in caged hens is one driving factor for the United States egg industry to explore options regarding alternative housing systems for laying hens. The aim of our research was to study the influence of housing systems on tibiae and humeri of 77-week-old Lohmann White hens. Pullets raised in an aviary system were either continued in aviary hen systems (AV) or conventional cages (AC) whereas pullets reared in conventional cages continued in conventional hen cages (CC) or enriched colony cages (EN) at 19 weeks. From each group, 120 hens were randomly euthanized and right and left tibae and humeri were excised for structural and mechanical analysis. Volumetric density of the cortical bone was measured using quantitative computed tomography (QCT). Aviary (AV) hens had greater cortical thickness and density but similar outer dimensions to AC hens (P Hens in EN system had humeri with similar cortical thickness and density but wider outer dimensions than the humeri of CC hens (P hens, whereas EN hens had denser tibial cortex than CC hens (P hens in the AV system were better able to protect their structure from endosteal resorption during the laying phase. Humeri of AV and EN hens had increased second moment of area compared to the AC and CC hens; however, the changes were not observed in tibiae. Mechanical property differences were observed, with bones of AV hens having greater failure moment and stiffness than AC hens and the same difference was observed between the EN and CC hens, (P hens.

  16. Electrophysiological properties of rat retinal Müller (glial) cells in postnatally developing and in pathologically altered retinae.

    Science.gov (United States)

    Felmy, F; Pannicke, T; Richt, J A; Reichenbach, A; Guenther, E

    2001-05-01

    Retinal glial Müller cells are characterized by dominant K(+) conductances. The cells may undergo changes of their membrane currents during ontogeny and gliosis as described in rabbit and man. Although the rat retina is often used in physiological experiments, the electrophysiology of rat Müller cells is less well studied. The aim of the present study was to characterize their membrane currents in postnatal development and in two models of retinal degeneration. Freshly isolated cells were subjected to whole-cell patch clamp recordings. During the first 4 weeks after birth of rats, their Müller cells displayed an increase in all membrane currents, particularly in the inward currents elicited at hyperpolarizing potentials. The decrease of the membrane resistance from more than 760 MOmega to less than 50 MOmega was accompanied by a shift of the zero current potential from about -20 mV to -80 mV, similar as earlier observed in developing rabbit Müller cells. These developmental changes were found in pigmented Brown Norway rats as well as in rats with inherited retinal dystrophy (RCS rats). Moreover, an infection of Lewis rats with the Borna disease virus caused substantial neuroretinal degeneration but did not result in a strong reduction of inward currents and of the zero current potential of the Müller cells. Thus, rat Müller cells fail to change their basic membrane properties in two different models of retinal pathology. This is in contrast to human and rabbit Müller cells, which have been shown to undergo dramatic changes of their membrane physiology in response to retinal diseases and injuries.

  17. Starch with a slow digestion property produced by altering its chain length, branch density, and crystalline structure.

    Science.gov (United States)

    Ao, Zihua; Simsek, Senay; Zhang, Genyi; Venkatachalam, Mahesh; Reuhs, Bradley L; Hamaker, Bruce R

    2007-05-30

    The hypothesis of increasing the branch density of starch to reduce its digestion rate through partial shortening of amylopectin exterior chains and the length of amylose was investigated. Starch products prepared using beta-amylase, beta-amylase and transglucosidase, maltogenic alpha-amylase, and maltogenic alpha-amylase and transglucosidase showed significant reduction of rapidly digested starch by 14.5%, 29.0%, 19.8%, and 31.0% with a concomitant increase of slowly digested starch by 9.0%, 19.7%, 5.7%, and 11.0%, respectively. The resistant starch content increased from 5.1% to 13.5% in treated starches. The total contents of the prebiotics isomaltose, isomaltotriose, and panose (Isomaltooligosaccharides) were 2.3% and 5.5%, respectively, for beta-amylase/transglucosidase- and maltogenic alpha-amylase/transglucosidase-treated starches. The molecular weight distribution of enzyme-treated starches and their debranched chain length distributions, analyzed using high-performance size-exclusion chromatography with multiangle laser light scattering and refractive index detection (HPSEC-MALLS-RI) and HPSEC-RI, showed distinctly different patterns among starches with different enzyme treatments. A larger proportion of low molecular weight fractions appeared in starches treated additionally with transglucosidase. All enzyme-treated starches showed a mixture of B- and V-type X-ray diffraction patterns, and 1H NMR spectra showed a significant increase of alpha-1,6 linkages. Both the increase of the starch branch density and the crystalline structure in the treated starches likely contribute to their slow digestion property.

  18. Comparison of physical and photophysical properties of monometallic and bimetallic ruthenium(II) complexes containing structurally altered diimine ligands

    Energy Technology Data Exchange (ETDEWEB)

    Macatangay, A.; Jackman, D.C.; Merkert, J.W. [Univ. of North Carolina, Charlotte, NC (United States)] [and others

    1996-11-06

    The physical and photophysical properties of a series of monometallic, [Ru(bpy){sub 2}(dmb)]{sup 2+}, [Ru(bpy){sub 2}(BPY)]{sup 2+}, [Ru(bpy)(Obpy)]{sup 2+} and [Ru(bpy){sub 2}(Obpy)] {sup 2+}, and bimetallic, [(Ru(bpy){sub 2}){sub 2}(BPY)]{sup 4+} and [(Ru(bpy){sub 2}){sub 2}(Obpy)]{sup 4+}, complexes are examined, where bpy is 2,2{prime}-bipyridine, dmb is 4,4{prime}-dimethyl-2,2{prime}-bipyridine, BPY is 1,2-bis(4-methyl-2,2{prime}-bipyridin-4{prime}-yl)ethane, and Obpy is 1,2-bis(2,2{prime}-bipyridin-6-yl)ethane. The complexes display metal-to-ligand charge transfer transitions in the 450 nmn region, intraligand {pi}{yields}{pi}* transitions at energies greater than 300 nm, a reversible oxidation of the ruthenium(II) center in the 1.25-1.40 V vs SSCE region, a series of three reductions associated with each coordinated ligand commencing at {minus}1.3 V and ending at {approximately}{minus}1.9 V, and emission from a {sup 3}MLCT state having energy maxima between 598 and 610 nm. The Ru{sup III}/Ru{sup II} oxidation of the two bimetallic complexes is a single, two one-electron process. Relative to [Ru(bpy){sub 2}(BPY)]{sup 2+}, the Ru{sup III}/Ru{sup II} potential for [Ru-(bpy){sub 2}(Obpy)]{sup 2+} increases from 1.24 to 1.35 V, the room temperature emission lifetime decreases from 740 to 3ns, and the emission quantum yield decreases from 0.078 to 0.000 23. Similarly, relative to [(Ru(bpy){sub 2}){sub 2}(BPY)]{sup 4+}, the Ru{sup III}/Ru{sup II} potential for [(Ru(bpy){sub 2}){sub 2}(Obpy)]{sup 4+} increases from 1.28 to 1.32 V, the room temperature emission lifetime decreases from 770 to 3 ns, and the room temperature emission quantum yield decreases from 0.079 to 0.000 26.

  19. Effects of fault-controlled CO2 alteration on mineralogical and geomechanical properties of reservoir and seal rocks, Crystal Geyser, Green River, Utah

    Science.gov (United States)

    Major, J. R.; Eichhubl, P.; Urquhart, A.; Dewers, T. A.

    2012-12-01

    An understanding of the coupled chemical and mechanical properties of reservoir and seal units undergoing CO2 injection is critical for modeling reservoir behavior in response to the introduction of CO2. The implementation of CO2 sequestration as a mitigation strategy for climate change requires extensive risk assessment that relies heavily on computer models of subsurface reservoirs. Numerical models are fundamentally limited by the quality and validity of their input parameters. Existing models generally lack constraints on diagenesis, failing to account for the coupled geochemical or geomechanical processes that affect reservoir and seal unit properties during and after CO2 injection. For example, carbonate dissolution or precipitation after injection of CO2 into subsurface brines may significantly alter the geomechanical properties of reservoir and seal units and thus lead to solution-enhancement or self-sealing of fractures. Acidified brines may erode and breach sealing units. In addition, subcritical fracture growth enhanced by the presence of CO2 could ultimately compromise the integrity of sealing units, or enhance permeability and porosity of the reservoir itself. Such unknown responses to the introduction of CO2 can be addressed by laboratory and field-based observations and measurements. Studies of natural analogs like Crystal Geyser, Utah are thus a critical part of CO2 sequestration research. The Little Grand Wash and Salt Wash fault systems near Green River, Utah, host many fossil and active CO2 seeps, including Crystal Geyser, serving as a faulted anticline CO2 reservoir analog. The site has been extensively studied for sequestration and reservoir applications, but less attention has been paid to the diagenetic and geomechanical aspects of the fault zone. XRD analysis of reservoir and sealing rocks collected along transects across the Little Grand Wash Fault reveal mineralogical trends in the Summerville Fm (a siltstone seal unit) with calcite and

  20. RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation.

    Science.gov (United States)

    Närvä, Elisa; Rahkonen, Nelly; Emani, Maheswara Reddy; Lund, Riikka; Pursiheimo, Juha-Pekka; Nästi, Juuso; Autio, Reija; Rasool, Omid; Denessiouk, Konstantin; Lähdesmäki, Harri; Rao, Anjana; Lahesmaa, Riitta

    2012-03-01

    Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied, the role of cytoplasmic regulators is still poorly characterized. Here, we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11, FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore, we demonstrate that OCT4, SOX2, and NANOG all bind to the promoter of L1TD1. Moreover, L1TD1 is highly expressed in seminomas, and depletion of L1TD1 in these cancer cells influences self-renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus, we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness.

  1. Expression of Arabidopsis glycine-rich RNA-binding protein AtGRP2 or AtGRP7 improves grain yield of rice (Oryza sativa) under drought stress conditions.

    Science.gov (United States)

    Yang, Deok Hee; Kwak, Kyung Jin; Kim, Min Kyung; Park, Su Jung; Yang, Kwang-Yeol; Kang, Hunseung

    2014-01-01

    Although posttranscriptional regulation of RNA metabolism is increasingly recognized as a key regulatory process in plant response to environmental stresses, reports demonstrating the importance of RNA metabolism control in crop improvement under adverse environmental stresses are severely limited. To investigate the potential use of RNA-binding proteins (RBPs) in developing stress-tolerant transgenic crops, we generated transgenic rice plants (Oryza sativa) that express Arabidopsis thaliana glycine-rich RBP (AtGRP) 2 or 7, which have been determined to harbor RNA chaperone activity and confer stress tolerance in Arabidopsis, and analyzed the response of the transgenic rice plants to abiotic stresses. AtGRP2- or AtGRP7-expressing transgenic rice plants displayed similar phenotypes comparable with the wild-type plants under high salt or cold stress conditions. By contrast, AtGRP2- or AtGRP7-expressing transgenic rice plants showed much higher recovery rates and grain yields compared with the wild-type plants under drought stress conditions. The higher grain yield of the transgenic rice plants was due to the increases in filled grain numbers per panicle. Collectively, the present results show the importance of posttranscriptional regulation of RNA metabolism in plant response to environmental stress and suggest that GRPs can be utilized to improve the yield potential of crops under stress conditions.

  2. The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains.

    Science.gov (United States)

    Sicard, H; Faubladier, M; Noaillac-Depeyre, J; Léger-Silvestre, I; Gas, N; Caizergues-Ferrer, M

    1998-08-01

    Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

  3. Thermal alteration of soil organic matter properties: a systematic study to infer response of Sierra Nevada climosequence soils to forest fires

    Science.gov (United States)

    Araya, Samuel N.; Fogel, Marilyn L.; Asefaw Berhe, Asmeret

    2017-02-01

    Fire is a major driver of soil organic matter (SOM) dynamics, and contemporary global climate change is changing global fire regimes. We conducted laboratory heating experiments on soils from five locations across the western Sierra Nevada climosequence to investigate thermal alteration of SOM properties and determine temperature thresholds for major shifts in SOM properties. Topsoils (0 to 5 cm depth) were exposed to a range of temperatures that are expected during prescribed and wild fires (150, 250, 350, 450, 550, and 650 °C). With increase in temperature, we found that the concentrations of carbon (C) and nitrogen (N) decreased in a similar pattern among all five soils that varied considerably in their original SOM concentrations and mineralogies. Soils were separated into discrete size classes by dry sieving. The C and N concentrations in the larger aggregate size fractions (2-0.25 mm) decreased with an increase in temperature, so that at 450 °C the remaining C and N were almost entirely associated with the smaller aggregate size fractions ( physical, chemical, elemental, and isotopic changes occurred at the mid-intensity fire temperatures, i.e., 350 and 450 °C. The magnitude of the observed changes in SOM composition and distribution in three aggregate size classes, as well as the temperature thresholds for critical changes in physical and chemical properties of soils (such as specific surface area, pH, cation exchange capacity), suggest that transformation and loss of SOM are the principal responses in heated soils. Findings from this systematic investigation of soil and SOM response to heating are critical for predicting how soils are likely to be affected by future climate and fire regimes.

  4. ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling.

    Directory of Open Access Journals (Sweden)

    Erhong Meng

    Full Text Available OBJECTIVE: Aldehyde dehydrogenase (ALDH expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. METHODS: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780 and platinum resistant (A2780/CP70 as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. RESULTS: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01. ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ and replication checkpoint (pS317 Chk1 were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. CONCLUSION: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

  5. Alterations of mass density and 3D osteocyte lacunar properties in bisphosphonate-related osteonecrotic human jaw bone, a synchrotron µCT study.

    Directory of Open Access Journals (Sweden)

    Bernhard Hesse

    Full Text Available Osteonecrosis of the jaw, in association with bisphosphonates (BRONJ used for treating osteoporosis or cancer, is a severe and most often irreversible side effect whose underlying pathophysiological mechanisms remain largely unknown. Osteocytes are involved in bone remodeling and mineralization where they orchestrate the delicate equilibrium between osteoclast and osteoblast activity and through the active process called osteocytic osteolysis. Here, we hypothesized that (i changes of the mineralized tissue matrix play a substantial role in the pathogenesis of BRONJ, and (ii the osteocyte lacunar morphology is altered in BRONJ. Synchrotron µCT with phase contrast is an appropriate tool for assessing both the 3D morphology of the osteocyte lacunae and the bone matrix mass density. Here, we used this technique to investigate the mass density distribution and 3D osteocyte lacunar properties at the sub-micrometer scale in human bone samples from the jaw, femur and tibia. First, we compared healthy human jaw bone to human tibia and femur in order to assess the specific differences and address potential explanations of why the jaw bone is exclusively targeted by the necrosis as a side effect of BP treatment. Second, we investigated the differences between BRONJ and control jaw bone samples to detect potential differences which could aid an improved understanding of the course of BRONJ. We found that the apparent mass density of jaw bone was significantly smaller compared to that of tibia, consistent with a higher bone turnover in the jaw bone. The variance of the lacunar volume distribution was significantly different depending on the anatomical site. The comparison between BRONJ and control jaw specimens revealed no significant increase in mineralization after BP. We found a significant decrease in osteocyte-lacunar density in the BRONJ group compared to the control jaw. Interestingly, the osteocyte-lacunar volume distribution was not altered after

  6. Thermal alteration of soil physico-chemical properties: a systematic study to infer response of Sierra Nevada climosequence soils to forest fires

    Science.gov (United States)

    Araya, Samuel N.; Meding, Mercer; Asefaw Berhe, Asmeret

    2016-07-01

    Fire is a common ecosystem perturbation that affects many soil properties. As global fire regimes continue to change with climate change, we investigated thermal alteration of soils' physical and chemical properties after they are exposed to a range of temperatures that are expected during prescribed and wildland fires. For this study, we used topsoils collected from a climosequence transect along the western slope of the Sierra Nevada that spans from 210 to 2865 m a.s.l. All the soils we studied were formed on a granitic parent material and had significant differences in soil organic matter (SOM) concentration and mineralogy owing to the effects of climate on soil development. Topsoils (0-5 cm depth) from the Sierra Nevada climosequence were heated in a muffle furnace at six set temperatures that cover the range of major fire intensity classes (150, 250, 350, 450, 550 and 650 °C). We determined the effects of heating temperature on soil aggregate strength, aggregate size distribution, specific surface area (SSA), mineralogy, pH, cation exchange capacity (CEC), and carbon (C) and nitrogen (N) concentrations. With increasing temperature, we found significant reduction of total C, N and CEC. Aggregate strength also decreased with further implications for loss of C protected inside aggregates. Soil pH and SSA increased with temperature. Most of the statistically significant changes (p soils that developed under different climate regimes. Our findings will be of interest to studies of inferences for how soils are likely to respond to different fire intensities under anticipated climate change scenarios.

  7. Correlation of wireline log characteristics with hydrothermal alteration and other reservoir properties of the Salton Sea and Westmorland geothermal fields, Imperial Valley, California, USA

    Energy Technology Data Exchange (ETDEWEB)

    Muramoto, F.S.; Elders, W.A.

    1984-05-01

    A detailed study of wireline logs from 11 wells in the Salton Sea and Westmorland geothermal systems was undertaken in order to determine the effects of hydrothermal alteration on the response of electrical and gamma-gamma density well logs. For the Salton Sea geothermal field, definite correspondence between log responses and hydrothermal mineralogy is evident, which in turn is related to the physical properties of the rocks. Three hydrothermal and one unaltered zone can be identified from log data on shales. These are: (1) the unaltered montmorillonite zone (<100/sup 0/ to 190/sup 0/C); (2) the illite zone (100/sup 0/ to 190/sup 0/C to 230/sup 0/ to 250/sup 0/C); (3) the chlorite zone (230/sup 0/ to 250/sup 0/C to 290/sup 0/ to 300/sup 0/C); and (4) the feldspar zone (>290/sup 0/ to 300/sup 0/C). The characteristic responses on well logs by which these zones are identified result primarily from changes in clay mineralogy of the shales and increases in density with progressive hydrothermal metamorphism. In the Westmorland geothermal field, differentiating mineral zones from log responses was only partially successful. However, analyses of both well log and petrologic data for wells Landers 1 and Kalin Farms 1 suggest that the former is heating up and the latter is cooling.

  8. Novel RNA-binding protein P311 binds eukaryotic translation initiation factor 3 subunit b (eIF3b) to promote translation of transforming growth factor β1-3 (TGF-β1-3).

    Science.gov (United States)

    Yue, Michael M; Lv, Kaosheng; Meredith, Stephen C; Martindale, Jennifer L; Gorospe, Myriam; Schuger, Lucia

    2014-12-05

    P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-β1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a Kd of 1.26 μm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-β 5'UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-βs 5'UTRs and eIF3b, stimulates the translation of TGF-β1, 2, and 3.

  9. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    Science.gov (United States)

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  10. BRCA-mutated Invasive Breast Carcinomas: Immunohistochemical Analysis of Insulin-like Growth Factor II mRNA-binding Protein (IMP3), Cytokeratin 8/18, and Cytokeratin 14.

    Science.gov (United States)

    Mohanty, Sambit K; Lai, Jin-Ping; Gordon, Ora K; Pradhan, Dinesh; Bose, Shikha; Dadmanesh, Farnaz

    2015-01-01

    To evaluate the expression of insulin-like growth factor II mRNA-binding protein (IMP3), CK8/18, and CK14 in BRCA mutated and sporadic invasive breast carcinoma. Immunohistochemistry for IMP3, CK8/18, and CK14 was performed on 39 cases of invasive breast carcinomas with BRCA mutation (24 BRCA1, 14 BRCA2, and 1 dual BRCA1/BRCA2) and 54 cases of sporadic invasive breast carcinomas. The relationship between the IMP3, CK8/18, and CK14 and the tumor grade and molecular phenotypes were analyzed. IMP3, CK8/18, and CK14 positivity were present in 20 (51%), 22 (56%), and 14 (36%) of 39 BRCA-mutated breast carcinomas, and 11 (20%), 53 (98%), and 24 (44%) of 54 sporadic breast carcinomas respectively. The rates of IMP3 expression and absence of CK8/18 (44% versus 2%) in BRCA-mutated breast carcinomas was significantly higher than the sporadic breast carcinomas (p = 0.002 and p BRCA1-related and BRCA2-related breast carcinomas in the immunoprofile for IMP3, CK8/18, and CK14. No significant correlation was identified between the expression of IMP3 and CK8/18 and the tumor grade in both BRCA-mutated and sporadic breast carcinomas (p > 0.05). In cases with luminal A and B phenotypes, the rates of expression of IMP3 and loss of CK8/18 were significantly higher in BRCA-mutated as compared to sporadic breast carcinoma (p BRCA-mutated breast carcinomas (54% versus 0%, p = 0.001), while no difference was observed for IMP3 expression (p = 0.435). Regardless of mutation type, histologic grade, or molecular phenotype, the absence of CK8/18 expression and presence of IMP3 expression are seen at much higher rate in BRCA mutated breast carcinomas.

  11. The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

    Science.gov (United States)

    Mobarak, C D; Anderson, K D; Morin, M; Beckel-Mitchener, A; Rogers, S L; Furneaux, H; King, P; Perrone-Bizzozero, N I

    2000-09-01

    The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

  12. Unfavorably Altered Fibrin Clot Properties in Patients with Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome: Association with Thrombin Generation and Eosinophilia.

    Directory of Open Access Journals (Sweden)

    Lucyna Mastalerz

    Full Text Available Given reports on the increased prevalence of thromboembolic incidents in patients with eosinophilic granulomatosis with polyangiitis (EGPA; Churg-Strauss syndrome, we investigated whether fibrin clot properties are unfavorably altered in EGPA.Ex vivo plasma fibrin clot characteristics, including clot permeability, turbidimetry and efficiency of fibrinolysis using two assays, were investigated in 34 consecutive patients with remission in EGPA according to the Birmingham Vasculitis Activity Score version 3 (23 female, 11 male, aged 48 (range, 21-80 years. The control group comprised 34 age- and sex- matched volunteers.Compared with controls, patients with EGPA were characterized by denser fiber clots (estimated pore size, Ks, 7.30±0.93 vs 10.14±1.07 10-9 cm2, faster fibrin polymerization (lag phase in a turbidimetric curve, 41.8±3.6 vs 47.4±2.9 s, thicker fibrin fibers (maximum absorbance, ΔAbs, 0.87±0.09 vs 0.72±0.07, higher maximum levels of D-dimer released from clots (DDmax 4.10±0.46 vs 3.54±0.35 mg/L, and prolonged clot lysis time (t50%; 9.50±1.45 vs 7.56±0.87 min; all p<0.0001. Scanning electron microscopy images confirmed denser plasma fibrin networks composed of thinner fibers formed in EGPA. Antineutrophil cytoplasmic antibody status and C-reactive protein did not affect clot variables. Multivariate analysis adjusted for fibrinogen showed that Ks was predicted by eosinophil count, peak thrombin generation, factor VIII, and soluble CD40 ligand, whereas eosinophil count, peak thrombin generation and antiplasmin predicted t50%.This study is the first to show that EGPA is associated with prothrombotic plasma fibrin clot phenotype, which may contribute to thromboembolic manifestations reported in this disease.

  13. Mutation I136V alters electrophysiological properties of the NaV1.7 channel in a family with onset of erythromelalgia in the second decade

    Directory of Open Access Journals (Sweden)

    Dib-Hajj Sulayman D

    2008-01-01

    Full Text Available Abstract Background Primary erythromelalgia is an autosomal dominant pain disorder characterized by burning pain and skin redness in the extremities, with onset of symptoms during the first decade in the families whose mutations have been physiologically studied to date. Several mutations of voltage-gated Na+ channel NaV1.7 have been linked with primary erythromelalgia. Recently, a new substitution NaV1.7/I136V has been reported in a Taiwanese family, in which pain appeared at later ages (9–22 years, with onset at 17 years of age or later in 5 of 7 family members, with relatively slow progression (8–10 years to involvement of the hands. The proband reported onset of symptoms first in his feet at the age of 11, which then progressed to his hands at the age of 19. The new mutation is located in transmembrane segment 1 (S1 of domain I (DI in contrast to all NaV1.7 mutations reported to date, which have been localized in the voltage sensor S4, the linker joining segments S4 and S5 or pore-lining segments S5 and S6 in DI, II and III. Results In this study, we characterized the gating and kinetic properties of I136V mutant channels in HEK293 cells using whole-cell patch clamp. I136V shifts the voltage-dependence of activation by -5.7 mV, a smaller shift in activation than the other erythromelalgia mutations that have been characterized. I136V also decreases the deactivation rate, and generates larger ramp currents. Conclusion The I136V substitution in NaV1.7 alters channel gating and kinetic properties. Each of these changes may contribute to increased excitability of nociceptive dorsal root ganglion neurons, which underlies pain in erythromelalgia. The smaller shift in voltage-dependence of activation of NaV1.7, compared to the other reported cases of inherited erythromelalgia, may contribute to the later age of onset and slower progression of the symptoms reported in association with this mutation.

  14. Attention Alters Perceived Attractiveness.

    Science.gov (United States)

    Störmer, Viola S; Alvarez, George A

    2016-04-01

    Can attention alter the impression of a face? Previous studies showed that attention modulates the appearance of lower-level visual features. For instance, attention can make a simple stimulus appear to have higher contrast than it actually does. We tested whether attention can also alter the perception of a higher-order property-namely, facial attractiveness. We asked participants to judge the relative attractiveness of two faces after summoning their attention to one of the faces using a briefly presented visual cue. Across trials, participants judged the attended face to be more attractive than the same face when it was unattended. This effect was not due to decision or response biases, but rather was due to changes in perceptual processing of the faces. These results show that attention alters perceived facial attractiveness, and broadly demonstrate that attention can influence higher-level perception and may affect people's initial impressions of one another.

  15. Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy.

    Science.gov (United States)

    Kowalik, Magdalena K; Rekawiecki, Robert; Kotwica, Jan

    2014-11-01

    The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5-6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.

  16. From Injectivity to Integrity Studies of CO{sub 2} Geological Storage Chemical Alteration Effects on Carbonates Petrophysical and Geomechanical Properties; Caracterisation de l'injectivite et de l'integrite d'un stockage geologique de CO{sub 2}: Effets d'une alteration chimique sur les proprietes petrophysiques et geomecaniques des roches carbonatees

    Energy Technology Data Exchange (ETDEWEB)

    Bemer, E.; Lombard, J.M. [Institut francais du petrole, IFP, 92 - Rueil-Malmaison (France)

    2010-05-15

    The technical and economical success of a CO{sub 2} geological storage project requires the preservation of the site injectivity and integrity properties over its lifetime. Unlike conventional hydrocarbon gas injection, CO{sub 2} injection implies geochemical reactions between the reactive brine and the in situ formations (reservoir and cap rock) leading to modifications of their petrophysical and geomechanical properties. This paper underlines the experimental difficulties raised by the low permeability of samples representative either of the cap rock itself or at least of transition zones between the reservoir and the effective cap rock. Acidification effects induced by CO{sub 2} injection have been studied using an experimental procedure of chemical alteration, which ensures a homogeneous dissolution pattern throughout the rock sample and especially avoids any worm-holing process that would lead to erroneous measurements at the core scale. Porosity, permeability and geomechanical properties of outcrop and field carbonate samples of various permeability levels have been measured under their native state and different levels of alteration. The present work has been conducted within the framework of ANR GeoCarbone-INJECTIVITY and GeoCarbone-INTEGRITY projects. Each experimental step: chemical alteration, petrophysical measurements and geomechanical testing, is considered from the point of view of injectivity and integrity issues. The obtained experimental data show clear trends of chemically induced mechanical weakening. (authors)

  17. Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks

    Science.gov (United States)

    Sebestyén, Endre; Singh, Babita; Miñana, Belén; Pagès, Amadís; Mateo, Francesca; Pujana, Miguel Angel; Valcárcel, Juan; Eyras, Eduardo

    2016-01-01

    Alternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number, and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells. These patterns are predicted to be mainly controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1. We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in nontumorigenic mammary epithelial cells. Our study uncovers novel splicing networks that potentially contribute to cancer development and progression. PMID:27197215

  18. The role of RNA-binding factor AUF1 in regulated gene expression and modulation of tumorigenesis%RNA 结合因子 AUF1调控基因表达及其在肿瘤发生中的双重调控作用的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨颖卓; 康鹏; 高杰; 徐春林; 王诗美(综述); 吴霞(审校)

    2014-01-01

    Turn-over of messenger ribonucleic acid ( mRNA) is a major control point in gene expres-sion.In mammals,many mRNAs encode inflammatory cytokines ,oncoproteins,and G-protein-coupled receptors are destabilized by the presence of AU -rich elements ( AREs ) in their 3′-untranslated regions .Association of ARE-binding proteins(AUBPs)with these mRNAs promotes rapid mRNA degradation .ARE/poly(U)-binding factor 1(AUF1),one of the best-characterized AUBPs,binds to many ARE-mRNAs and assembles other fac-tors to recruit the mRNA degradation machinery .Most studies support an mRNA -destabilizing role for AUF1,al-though other findings suggest additional functions for this factor .However,several lines of evidence also support a role for AUF1 in the initiation and/or development of cancer .Many AUF1-targeted transcripts encode products that control pro-or anti-oncogenic processes .Numerous signaling pathways alter the composition of this AUF 1 complex of proteins to affect changes in ARE -mRNA degradation rates .This review briefly describes the roles of mRNA decay in gene expression in general and ARE -mediated decay ( AMD) in particular ,with a focus on AUF1 and the different modes of regulation that govern AUF 1 involvement in AMD.In the end,we discuss how changes in AUF1 isoform distribution,subcellular localization,and post-translational protein modifications can influence the metabolism of targeted mRNAs .%信使RNA( Messenger ribonucleic acid ,mRNA)不断更新在基因表达中具有重要作用,而且编码一些重要蛋白。本文详细阐述了RNA结合因子1(RNA-binding factor 1,AUF1)使mRNA稳定或去稳定,在转录后水平调控基因表达的可能的分子机制,归纳了AUF1各个亚型蛋白在基因表达中的独特作用的相关研究进展,并探讨了AUF1表达水平的改变在肿瘤发生中的双重调控作用。认识到AUF1发挥生物学效应的分子机制以及作用的具体信号通路,能够帮助我们揭开更多的生命谜题。

  19. Artificial cold exposure induced stroke in renovascular hypertensive rats and its association with cold-inducible RNA binding protein mRNA expression in brain tissue and blood pressure

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: High incidence of stroke at interchange period of autumn and winter was demonstrated by epidemiological survey, and the specific causes should be further investigated.OBJECTIVE: To investigate the influence of artificial cold exposure on the incidence of stroke in renovascular hypertensive rats (RHR), and analyze the association with blood pressure and cold-inducible RNA binding protein (CIRP) mRNA expression in brain tissue.DESIGN: A completely randomized grouping design, a randomized control animal trial.SETTINGS: Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University; Department of Chemistry, Open laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong.MATERIALS: Male SD rats (n =460), weighing 80- 100 g were obtained from Guangdong Province Health Animal Unit. A modified RXZ-300A intelligent artificial climate cabinet (Ningbo Jiangnan Instrument Co.,Ltd., China).METHODS: The experiment were processed in the Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University and the Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong from October 2004 to November 2005. Rats (n =400) were operated to establish 2-kidney 2-clip RHR model as described previously. The sham-operated rats (n =60) served as normotensive controls. Eight weeks later, 300 of RHR were randomly selected according to their systolic blood pressure (SBP) and divided into 3 sub-groups (n =100 per group): mild hypertensive group (SBP of 160 - 200 mm Hg), moderate hypertensive group (SBP of 200 - 220 mm Hg) and severe hypertensive group (SBP > 220 mm Hg). Each group was further divided into two groups (n =50) under ACE and non-ACE. Normal sham-operated SD rats (n =60), SBP < 140 mm Hg, were randomly divided into two groups: Sham-operated control group (n =30) under ACE and non-ACE. To establish the ACE and non

  20. Clone and analysis of human RNA binding protein with multiple splicing 2 gene%人类多重剪接RNA结合蛋白类基因的克隆和分析

    Institute of Scientific and Technical Information of China (English)

    张雅娟; 肖娟娟; 鲁德意; 王尉平

    2013-01-01

    Objective To assemble and clone human RNA binding protein with multiple splicing 2(RBPMS2) gene by homologous sequence aligning. Methods Human RBPMS2 gene was taken as an information probe for searching the coding sequece in GeneBank database by homologous sequence aligning. Then the highly homologous expressed sequence tag(EST) was assembled to the contig using VECTOR NTI software. The chromosome localization,domain and expression pattern of RBPMS2 were analyzed by UCSC Genome Blat Server, SMART and Unigene database, respectively. Results A novel cDNA sequence of human RBPMS2 gene was cloned, which contained complete open reading frame(ORF). RBPMS2 gene encoded a 209 amino acides protein with MW 22. 5 KDa and pi 8.63. SMART analysis showed that RBPMS2 protein included a RNA recognition motif (RRM) domain. RBPMS2 was mapping on the fifteenth chromosome, located at 15q22. 31, and consisted of seven exons and six introns. Unigene database presented that RBPMS2 was widely expressed in ovotid,zygote,blastula,embryo at different developmental stages, bladder and kidney. Conclusion A novel human RBPMS2 gene has been cloned and analyzed.%目的 通过同源筛选的方法拼接并克隆人类多重剪接RNA结合蛋白2(RBPMS2)基因.方法 采用同源筛选策略,以人RBPMS2基因作为信息探针,在GeneBank数据库中进行分析,将获得的高度同源的表达序列标签(EST)用VECTOR NTI软件拼接成重叠群.通过UCSCGenome Blat Server分析,确定RBPMS2基因的染色体定位,SMART网上分析工具进行结构域预测,Unigene数据库进行表达谱分析.结果 电子克隆到人类RBPMS2新基因cDNA序列,含完整的开放阅读框(ORF).RBPMS2基因编码的多肽由209个氨基酸组成,分子量22.5 KDa,等电点8.63.SMART分析显示RBPMS2蛋白包含1个RNA识别模体(RRM)结构域.RBPMS2基因位于第15号染色体,定位在15q22.31,由7个外显子和6个内含子组成.电子表达谱分析显示RBPMS2在卵细胞

  1. Lignin properties in topsoils of a beech/oak forest after 8 years of manipulated litter fall: relevance of altered input and oxidation of lignin

    NARCIS (Netherlands)

    T. Klotzbücher; S. Strohmeier; K. Kaiser; R.D. Bowden; K. Lajtha; H. Ohm; K. Kalbitz

    2013-01-01

    Background and aims We studied the response of lignin oxidation in soils of a beech/oak forest to changes in litter fall. Additionally we considered possible factors in lignin oxidation, including altered (i) input of fresh organic matter and (ii) fungi-to-bacteria ratios. Methods The field-based ex

  2. Reversible Li-insertion in nanoscaffolds: A promising strategy to alter the hydrogen sorption properties of Li-based complex hydrides

    NARCIS (Netherlands)

    Ngene, Peter; Verkuijlen, Margriet H. W.; Barre, Charlotte; Kentgens, Arno P. M.; de Jongh, Petra E.

    2016-01-01

    Intercalation and de-intercalation of lithium into graphene layers is a well-established phenomenon in Li-ion battery technology. Here we show how this phenomenon can be exploited to destabilize, and alter the hydrogen sorption behaviour of Li-based metal hydrides (LiBH4 and LiAlH4), thereby achievi

  3. Insights into magmatic processes and hydrothermal alteration of in situ superfast spreading ocean crust at ODP/IODP site 1256 from a cluster analysis of rock magnetic properties

    NARCIS (Netherlands)

    Dekkers, Mark J.; Heslop, David; Herrero-Bervera, Emilio; Acton, Gary; Krasa, David

    2014-01-01

    We analyze magnetic properties from Ocean Drilling Program (ODP)/Integrated ODP (IODP) Hole 1256D (6°44.1' N, 91°56.1' W) on the Cocos Plate in ∼15.2 Ma oceanic crust generated by superfast seafloor spreading, the only drill hole that has sampled all three oceanic crust layers in a tectonically undi

  4. Regularity of the Damage of Altered Rock’ s Mechanical Properties Under Water-Rock Interaction%水岩作用下蚀变岩力学性质损伤规律

    Institute of Scientific and Technical Information of China (English)

    牛传星; 秦哲; 冯佰研; 付厚利

    2016-01-01

    In order to research the effect of water level fluctuation on the mechanical properties of slope rock at al -tered zones , we carried out uniaxial compression test on rock samples from open-pit slope in Cangshang under dif-ferent saturation-dehydration cycles .Moreover , we established the function which describes the variation pattern of mechanical properties of altered rock under water-rock interaction .We also analyzed the influence of circulating times on the mechanical properties of altered rock through introducing the concept of damage rate .Besides , the damage mechanism of water-rock interaction on altered rocks was also analyzed by scanning electron microscopy ( SEM) .The result indicated that the mechanical properties of altered rocks were obviously weakened by water -rock interaction , and with the increase of water-rock interaction , the phenomenon was more obvious;with the increase of circulating times, the elasticity modulus decayed rapidly while the peak intensity was decaying continuously .As the degree of alteration and fragmentation increased , the degree of damage increased regarding the water-rock interac-tion to the mechanical properties of rock , and after fifteen saturation-dehydration cycles , the damage rate of elastic-ity modulus reached 60%, while that of peak intensity was 45%.%为了研究水位升降对蚀变带边坡岩石力学性质的影响,选取仓上露天金矿坑边坡蚀变岩样,进行了不同饱水-失水循环次数的单轴压缩试验。建立了函数关系描述蚀变岩力学性质在水岩作用下的变化规律,引入损伤率的概念分析了循环次数对蚀变岩力学性质的影响,并利用电镜扫描技术从微观角度分析了水岩作用对蚀变岩的损伤机理。结果表明:水岩作用对蚀变岩的力学性质有明显的弱化现象,且水岩作用越强,弱化现象越明显;不同循环次数下,蚀变岩弹性模量随循环次数增加衰减迅速,峰值强度呈

  5. The single-channel properties of human acetylcholine α7 receptors are altered by fusing α7 to the green fluorescent protein

    Science.gov (United States)

    Fucile, Sergio; Palma, Eleonora; Martínez-Torres, Ataúlfo; Miledi, Ricardo; Eusebi, Fabrizio

    2002-01-01

    Neuronal nicotinic acetylcholine (AcCho) receptors composed of α7-subunits (α7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) α7-AcChoRs, we identified two classes of channel conductance: a low conductance (γL) of 72 pS and a high one (γH) of 87 pS, with mean open-times (τop) of 0.6 ms. The same classes of conductances, but longer τop (3 ms), were seen in experiments with chimeric α7 receptors in which the wtα7 extracellular C terminus was fused to the green fluorescent protein (wtα7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing α7 receptors carrying a Leu-to-Thr 248 mutation (mutα7) or oocytes expressing chimeric mutα7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wtα7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter τop, than those activated by AcCho. Accordingly, human homomeric wtα7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mutα7 receptors open channels of lower conductance and longer lifetime than those gated by wtα7-AcChoRs, and these parameters are not greatly altered by fusing the mutα7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function. PMID:11891309

  6. Ethylmalonic encephalopathy ETHE1 R163W/R163Q mutations alter protein stability and redox properties of the iron centre.

    Directory of Open Access Journals (Sweden)

    Bárbara J Henriques

    Full Text Available ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE. Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association.

  7. Ethylmalonic encephalopathy ETHE1 R163W/R163Q mutations alter protein stability and redox properties of the iron centre.

    Science.gov (United States)

    Henriques, Bárbara J; Lucas, Tânia G; Rodrigues, João V; Frederiksen, Jane H; Teixeira, Miguel S; Tiranti, Valeria; Bross, Peter; Gomes, Cláudio M

    2014-01-01

    ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE). Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association.

  8. ErbB4基因micro-RNA靶序列单核苷酸多态性与乳腺癌易感性的关系%Relationship between polymorphism of ErbB4 gene in mirco-RNA binding site and the risk for breast cancer

    Institute of Scientific and Technical Information of China (English)

    朱晓灵; 宋丰举; 郑红; 张丽娜; 赵妍蕊; 陈可欣

    2011-01-01

    目的:探讨ErbB4基因micro-RNA靶序列单核苷酸多态性与乳腺癌易感性的关系.方法:采用TagMan单核苷酸多态(single nucleotide polymorphism,SNP)分型技术检测1 509例乳腺癌患者和1 517例健康对照者外周血中ErbB4基因micro-RNA靶序列rs1595066及rs16845990位点的基因型.结果:ErbB4基因micro-RNA靶序列rs16845990位点3种基因型在乳腺癌患者和健康对照者间的分布差异无统计学意义(P=0.302):rs1595066位点3种基因型在2组间的分布差异有统计学意义(P=0.045),与GG型相比,杂合型AG及纯合型AA均可显著降低乳腺癌的发病风险,调整后比值比(odds ratios,OR)及其95%可信区间(confidence intervals,CI)分别为0.81(0.69~0.95)和0.75 (0.58~0.96).分层分析显示,此保护作用在年龄≧55岁、无肿瘤家族史和无乳腺良性疾病史者中更显著.结论:ErbB4基因micro-RNA靶序列rs1595066 G>A位点A等位基因可能降低乳腺癌的发病风险.%Objective: To investigate the relationship between single nucleotide polymorphisrn (SNP) of ErbB4 gene in mirco-RNA binding site and the susceptibility of breast cancer. Methods: Genotypes of ErbB4 gene in mirco-RNA binding site at rs1595066 and rs16845990 were measured using the TaqMan SNP method in 1 509 patients with breast cancer and 1 517 healthy controls. Results: Three kinds of genotype of ErbB4 gene in mirco-RNA binding site at rs1595066 were significantly different between breast cancer patients and the healthy controls (P=0.045), while there was no difference at rs1 6845990 site (P=0.302). The carriers of AG and AA genotypes at rs1595065 site had lower risk of breast cancer than GG genotype [(odds ratio, OR) = 0.81, 95% (confidence interval, CI) = 0.69-0.95; OR = 0.75, 95% CI = 0.58-0.96]. Stratified analysis showed that this protective effect was significant in the subjects aged over 55 years, without a history of benign breast disease and without a family history of cancer. Conclusion: The

  9. Tuning the morphology, luminescence and magnetic properties of hexagonal-phase NaGdF4: Yb, Er nanocrystals via altering the addition sequence of the precursors

    Science.gov (United States)

    Zhao, Shuwen; Xia, Donglin; Zhao, Ruimin; Zhu, Hao; Zhu, Yiru; Xiong, Yuda; Wang, Youfa

    2017-01-01

    Hexagonal-phase NaGdF4: Yb, Er upconversion nanocrystals (UCNCs) with tunable morphology and properties were successfully prepared via a thermal decomposition method. The influences of the adding sequence of the precursors on the morphology, chemical composition, luminescence and magnetic properties were investigated by transmission electron microscopy (TEM), inductively coupled plasma-atomic emission spectrometry (ICP-AES), upconversion (UC) spectroscopy, and a vibrating sample magnetometer (VSM). It was found that the resulting nanocrystals, with different sizes ranging from 24 to 224 nm, are in the shape of spheres, hexagonal plates and flakes; moreover, the composition percentage of Yb3+-Er3+ and Gd3+ ions was found to vary in a regular pattern with the adding sequence. Furthermore, the intensity ratios of emission colors (f g/r, f g/p), and the magnetic mass susceptibility of hexagonal-phase NaGdF4: Yb, Er nanocrystals change along with the composition of the nanocrystals. A positive correlation between the susceptibility and f g/r of NaGdF4: Yb, Er was proposed. The decomposition processes of the precursors were investigated by a thermogravimetric (TG) analyzer. The result indicated that the decomposition of the resolved lanthanide trifluoroacetate is greatly different from lanthanide trifluoroacetate powder. It is of tremendous help to recognize the decomposition process of the precursors and to understand the related reaction mechanism.

  10. Leukocyte inclusion within a platelet rich plasma-derived fibrin scaffold stimulates a more pro-inflammatory environment and alters fibrin properties.

    Science.gov (United States)

    Anitua, Eduardo; Zalduendo, Mar; Troya, María; Padilla, Sabino; Orive, Gorka

    2015-01-01

    One of the main differences among platelet-rich plasma (PRP) products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF) and leukocyte-platelet rich plasma (L-PRP) scaffolds was determined by enzyme-linked immunosorbent assay (ELISA) and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions.

  11. Iceland as a Model for Chemical Alteration on Mars

    Science.gov (United States)

    Bishop, J. L.; Schiffman, P.; Murad, E.; Southard, R.

    2001-03-01

    Subglacial volcanic activity on Iceland has led to the formation of a variety of silicate and iron oxide-rich alteration products that may ressemble chemical alteration on Mars. The spectral and chemical properties of Icelandic samples are presented.

  12. Untangling Magmatic Processes and Hydrothermal Alteration of in situ Superfast Spreading Ocean Crust at ODP/IODP Site 1256 with Fuzzy c-means Cluster Analysis of Rock Magnetic Properties

    Science.gov (United States)

    Dekkers, M. J.; Heslop, D.; Herrero-Bervera, E.; Acton, G.; Krasa, D.

    2014-12-01

    Ocean Drilling Program (ODP)/Integrated ODP (IODP) Hole 1256D (6.44.1' N, 91.56.1' W) on the Cocos Plate occurs in 15.2 Ma oceanic crust generated by superfast seafloor spreading. Presently, it is the only drill hole that has sampled all three oceanic crust layers in a tectonically undisturbed setting. Here we interpret down-hole trends in several rock-magnetic parameters with fuzzy c-means cluster analysis, a multivariate statistical technique. The parameters include the magnetization ratio, the coercivity ratio, the coercive force, the low-field susceptibility, and the Curie temperature. By their combined, multivariate, analysis the effects of magmatic and hydrothermal processes can be evaluated. The optimal number of clusters - a key point in the analysis because there is no a priori information on this - was determined through a combination of approaches: by calculation of several cluster validity indices, by testing for coherent cluster distributions on non-linear-map plots, and importantly by testing for stability of the cluster solution from all possible starting points. Here, we consider a solution robust if the cluster allocation is independent of the starting configuration. The five-cluster solution appeared to be robust. Three clusters are distinguished in the extrusive segment of the Hole that express increasing hydrothermal alteration of the lavas. The sheeted dike and gabbro portions are characterized by two clusters, both with higher coercivities than in lava samples. Extensive alteration, however, can obliterate magnetic property differences between lavas, dikes, and gabbros. The imprint of thermochemical alteration on the iron-titanium oxides is only partially related to the porosity of the rocks. All clusters display rock magnetic characteristics in line with a stable NRM. This implies that the entire sampled sequence of ocean crust can contribute to marine magnetic anomalies. Determination of the absolute paleointensity with thermal techniques is

  13. Leukocyte inclusion within a platelet rich plasma-derived fibrin scaffold stimulates a more pro-inflammatory environment and alters fibrin properties.

    Directory of Open Access Journals (Sweden)

    Eduardo Anitua

    Full Text Available One of the main differences among platelet-rich plasma (PRP products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF and leukocyte-platelet rich plasma (L-PRP scaffolds was determined by enzyme-linked immunosorbent assay (ELISA and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions.

  14. Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

    Science.gov (United States)

    Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

    2016-04-15

    Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae.

  15. Long-lasting alterations in membrane properties, K+ currents and glutamatergic synaptic currents of nucleus accumbens medium spiny neurons in a rat model of alcohol dependence

    Directory of Open Access Journals (Sweden)

    Igor eSpigelman

    2012-06-01

    Full Text Available Chronic alcohol exposure causes marked changes in reinforcement mechanisms and motivational state that are thought to contribute to the development of cravings and relapse during protracted withdrawal. The nucleus accumbens (NAcc is a key structure of the mesolimbic dopaminergic reward system. Although the NAcc plays an important role in mediating alcohol-seeking behaviors, little is known about the molecular mechanisms underlying alcohol-induced neuroadaptive changes in NAcc function. The aim of this study was to investigate the effects of chronic intermittent ethanol (CIE treatment, a rat model of alcohol withdrawal and dependence, on intrinsic electrical membrane properties and glutamatergic synaptic transmission of medium spiny neurons (MSNs in the NAcc core during protracted withdrawal. We show that CIE treatment followed by prolonged withdrawal increased the inward rectification of MSNs observed at hyperpolarized potentials. In addition, MSNs from CIE-treated animals displayed a lower input resistance, faster action potentials (APs and larger fast afterhyperpolarizations (fAHPs than MSNs from vehicle-treated animals, all suggestive of increases in K+-channel conductances. Significant increases in the Cs+-sensitive inwardly-rectifying K+-current accounted for the increased input resistance, while increases in the A-type K+-current accounted for the faster APs and increased fAHPs in MSNs from CIE rats. We also show that the amplitude and the conductance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR-mediated mEPSCs were enhanced in CIE-treated animals due to an increase in a small fraction of functional postsynaptic GluA2-lacking AMPARs. These long-lasting modifications of excitability and excitatory synaptic receptor function of MSNs in the NAcc core could play a critical role in the neuroadaptive changes underlying alcohol withdrawal and dependence.

  16. Intermittent Theta-Burst Transcranial Magnetic Stimulation Alters Electrical Properties of Fast-Spiking Neocortical Interneurons in a