WorldWideScience

Sample records for ires div rna

  1. Structural and functional basis for RNA cleavage by Ire1

    Directory of Open Access Journals (Sweden)

    Stroud Robert M

    2011-07-01

    Full Text Available Abstract Background The unfolded protein response (UPR controls the protein folding capacity of the endoplasmic reticulum (ER. Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. Results This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. Conclusions Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

  2. Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

    Science.gov (United States)

    Khan, Mateen A; Ma, Jia; Walden, William E; Merrick, William C; Theil, Elizabeth C; Goss, Dixie J

    2014-06-01

    Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor

    KAUST Repository

    Nagashima, Yukihiro

    2011-07-01

    IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.

  4. Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

    Science.gov (United States)

    Colussi, Timothy M; Costantino, David A; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A; Jaafar, Zane A; Plank, Terra-Dawn M; Noller, Harry F; Kieft, Jeffrey S

    2015-03-05

    The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

  5. MicroRNA-134 regulates poliovirus replication by IRES targeting

    OpenAIRE

    Bakre, Abhijeet A.; Shim, Byoung-Shik; Tripp, Ralph A.

    2017-01-01

    Global poliovirus eradication efforts include high vaccination coverage with live oral polio vaccine (OPV), surveillance for acute flaccid paralysis, and OPV “mop-up” campaigns. An important objective involves host-directed strategies to reduce PV replication to diminish viral shedding in OPV recipients. In this study, we show that microRNA-134-5p (miR-134) can regulate Sabin-1 replication but not Sabin-2 or Sabin-3 via direct interaction with the PV 5′UTR. Hypochromicity data showed miR-134 ...

  6. HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

    Science.gov (United States)

    Filbin, Megan E; Kieft, Jeffrey S

    2011-07-01

    Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

  7. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

    Directory of Open Access Journals (Sweden)

    Marie-Jo Halaby

    2015-01-01

    Full Text Available Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES that is located at the 5′-untranslated region (UTR of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80 has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress.

  8. Ire1 mediated mRNA splicing in a C-terminus deletion mutant of Drosophila Xbp1.

    Directory of Open Access Journals (Sweden)

    Dina S Coelho

    Full Text Available The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions (the hydrophobic region 2--HR2--and the C-terminal translational pausing site present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. Here, we generated a Drosophila Xbp1 deletion mutant (Excision101 lacking both HR2 and C-terminal region, but not the Ire1 splicing site. We show that Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. Our results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced.

  9. Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

    International Nuclear Information System (INIS)

    Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

    2006-01-01

    The iron regulatory protein IRP1 has been crystallized in a complex with ferritin IRE RNA and a complete data set has been collected to 2.8 Å resolution. Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe–4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe–4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe–S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 Å, β = 92.0°. Native data sets have been collected from several crystals with resolution extending to 2.8 Å and the structure has been solved by molecular replacement

  10. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor

    KAUST Repository

    Nagashima, Yukihiro; Mishiba, Kei-ichiro; Suzuki, Eiji; Shimada, Yukihisa; Iwata, Yuji; Koizumi, Nozomu

    2011-01-01

    -type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated

  11. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    Directory of Open Access Journals (Sweden)

    Jae-Su Moon

    Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

  12. Divergent picornavirus IRES elements

    DEFF Research Database (Denmark)

    Belsham, Graham

    2009-01-01

    Internal ribosome entry site (IRES) elements were first identified about 20 years ago within the 5' untranslated region of picornavirus RNAs. They direct a cap-independent mechanism of translation initiation on the viral RNA. Within the picornavirus family it is now known that there are four...... classes of IRES element which vary in size (450-270nt), they also have different, complex, secondary structures and distinct requirements for cellular proteins to allow them to function. This review describes the features of each class of picornavirus IRES element but focuses on the characteristics...... of the most recently described group, initially identified within the porcine teschovirus-1 RNA, which has strong similarities to the IRES elements from within the genomes of hepatitis C virus and the pestiviruses which are members of the flavivirus family. The selection of the initiation codon...

  13. AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

    Science.gov (United States)

    Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

    2015-10-01

    Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein. © 2015 John Wiley & Sons Ltd.

  14. The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES.

    Science.gov (United States)

    Yu, Yingpu; Sweeney, Trevor R; Kafasla, Panagiota; Jackson, Richard J; Pestova, Tatyana V; Hellen, Christopher Ut

    2011-08-26

    Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G-interacting domain is structurally distinct, although it contains an apical eIF4G-interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract-binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin.

  15. Specificity in endoplasmic reticulum-stress signaling in yeast entails a step-wise engagement of HAC1 mRNA to clusters of the stress sensor Ire1.

    Science.gov (United States)

    van Anken, Eelco; Pincus, David; Coyle, Scott; Aragón, Tomás; Osman, Christof; Lari, Federica; Gómez Puerta, Silvia; Korennykh, Alexei V; Walter, Peter

    2014-12-30

    Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.

  16. Viral IRES prediction system - a web server for prediction of the IRES secondary structure in silico.

    Directory of Open Access Journals (Sweden)

    Jun-Jie Hong

    Full Text Available The internal ribosomal entry site (IRES functions as cap-independent translation initiation sites in eukaryotic cells. IRES elements have been applied as useful tools for bi-cistronic expression vectors. Current RNA structure prediction programs are unable to predict precisely the potential IRES element. We have designed a viral IRES prediction system (VIPS to perform the IRES secondary structure prediction. In order to obtain better results for the IRES prediction, the VIPS can evaluate and predict for all four different groups of IRESs with a higher accuracy. RNA secondary structure prediction, comparison, and pseudoknot prediction programs were implemented to form the three-stage procedure for the VIPS. The backbone of VIPS includes: the RNAL fold program, aimed to predict local RNA secondary structures by minimum free energy method; the RNA Align program, intended to compare predicted structures; and pknotsRG program, used to calculate the pseudoknot structure. VIPS was evaluated by using UTR database, IRES database and Virus database, and the accuracy rate of VIPS was assessed as 98.53%, 90.80%, 82.36% and 80.41% for IRES groups 1, 2, 3, and 4, respectively. This advance useful search approach for IRES structures will facilitate IRES related studies. The VIPS on-line website service is available at http://140.135.61.250/vips/.

  17. Insight into the recognition, binding, and reactivity of catalytic metallodrugs targeting stem loop IIb of hepatitis C IRES RNA.

    Science.gov (United States)

    Bradford, Seth S; Ross, Martin James; Fidai, Insiya; Cowan, James A

    2014-06-01

    The complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb (SLIIb) of the hepatitis C virus (HCV) internal ribosomal entry site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein we describe additional studies focused on understanding the cleavage mechanism as well as the relationship of catalyst configuration to structural recognition and site-selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI-TOF mass spectrometry, melting temperature determinations, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all-D-amino acid form of this metallopeptide, complex 2-Cu, are reported and compared with those of complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD value of 76 ± 3 nM, and Michaelis-Menten parameters of kcat =0.14 ± 0.01 min(-1) and KM =7.9 ± 1.2 μM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to that of the 1-Cu parent peptide, with an IC50 value of 1.9 ± 0.4 μM and cytotoxicity exceeding 100 μM. RT-PCR experiments confirm a significant decrease in HCV RNA levels in replicon assays for up to nine days when treated with complex 1-Cu in three-day dosing increments. This study shows the influence that the α-carbon stereocenter has for this new class of compounds, while detailed mass spectrometry and computational analyses provide new insight into the mechanisms of recognition, binding, and reactivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES.

    Science.gov (United States)

    Boehringer, Daniel; Thermann, Rolf; Ostareck-Lederer, Antje; Lewis, Joe D; Stark, Holger

    2005-11-01

    Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.

  19. Insights into the Biology of IRES Elements through Riboproteomic Approaches

    Directory of Open Access Journals (Sweden)

    Almudena Pacheco

    2010-01-01

    Full Text Available Translation initiation is a highly regulated process that exerts a strong influence on the posttranscriptional control of gene expression. Two alternative mechanisms govern translation initiation in eukaryotic mRNAs, the cap-dependent initiation mechanism operating in most mRNAs, and the internal ribosome entry site (IRES-dependent mechanism, first discovered in picornaviruses. IRES elements are highly structured RNA sequences that, in most instances, require specific proteins for recruitment of the translation machinery. Some of these proteins are eukaryotic initiation factors. In addition, RNA-binding proteins (RBPs play a key role in internal initiation control. RBPs are pivotal regulators of gene expression in response to numerous stresses, including virus infection. This review discusses recent advances on riboproteomic approaches to identify IRES transacting factors (ITAFs and the relationship between RNA-protein interaction and IRES activity, highlighting the most relevant features on picornavirus and hepatitis C virus IRESs.

  20. The IRES electronic seal

    International Nuclear Information System (INIS)

    Autrusson, B.; Brochard, D.; Moreau, J.F.; Martin, J.C.

    2001-01-01

    In the framework of the French Support Program for the IAEA Safeguards, the 'Institut de Protection et de Surete Nucleaire' (IPSN), developed an electronic seal called Integrated and Reusable Electronic Seal (IRES) that enables independent verification by different inspectorates (IAEA, Euratom, and National Inspectorate). The seal can be remotely interrogated by radio frequency and integrated to other Containment/surveillance systems by serial line RS 485. Data are authenticated and the IRESMAG software manages in the seal reader all functionalities of the seal and records inspection data compatible with the IAEA's Seal Database. To perform this development, IPSN relies on industrial partners: SAPHYMO for the general architecture of the seal and the electronics, THALES for the authentication of data and the security of transmission. The main features of the IRES seal are the following: Interrogation by different inspectorate, allowing independent conclusions; Recording of events, including tampering, in a non-volatile memory; Authentication of data and enhanced security of the communication between the seal and the seal reader; Remote interrogation by an inspector or/and automatic for unattended systems or remote monitoring; Reusable after erasing the seal memory and replacement of the batteries

  1. The IRES electronic seal

    International Nuclear Information System (INIS)

    Gourlez, P.; Funk, P.; Brochard, D.; Moreau, J.F.; Martin, J.C.

    2001-01-01

    In the framework of the French Support Program for the IAEA Safeguards, the 'Institut de Protection et de Surete Nucleaire' (IPSN), developed an electronic seal called Integrated and Reusable Electronic Seal (IRES) that enables independent verification by different inspectorates (IAEA, Euratom, and National Inspectorate) Furthermore, a bilateral co-ordination between Euratom and French domestic safeguards takes place in some French facilities regarding a common approach concerning the seals especially in case of crisis situation. The seal can be remotely interrogated by radio frequency and integrated to other Containment/surveillance systems by serial line RS 485. Data are authenticated and the IRESMAG software manages in the seal reader all functionalities of the seal and records inspection data compatible with the IAEA's Seal Database

  2. Structural Features of the Seneca Valley Virus Internal Ribosome Entry Site (IRES) Element: a Picornavirus with a Pestivirus-Like IRES

    DEFF Research Database (Denmark)

    Willcocks, Margaret M.; Locker, Nicolas; Gomwalk, Zarmwa

    2011-01-01

    The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the FLAVIVIRIDAE: The SVV IRES...... has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates...

  3. Activation of IRE1α-XBP1 pathway induces cell proliferation and invasion in colorectal carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Chun [Department of Coloproctology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000 (China); Jin, Zhao [Department of Coloproctology, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, Wenzhou 325000 (China); Chen, Nian-zhao [Department of Medicine, The Chinese Medicine Hospital of Wenzhou, Wenzhou 325000 (China); Lu, Min; Liu, Chang-bao; Hu, Wan-Le [Department of Coloproctology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000 (China); Zheng, Chen-guo, E-mail: zhengchenguo80@163.com [Department of Coloproctology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000 (China)

    2016-01-29

    Cell proliferation and tumor metastasis are considered as the main reasons for death in colorectal carcinoma (CRC). IRE1α-XBP1 pathway is the most conserved UPR pathways, which are activated during ER stress caused by the accumulation of unfolded or misfolded protein in the lumen of ER. Here, we demonstrated the critical role of IRE1α-XBP1 pathway and underlying molecular mechanism in cell proliferation and tumor metastasis in CRC. By the use of tissue microarray analysis of samples from 119 patients with CRC, IRE1α was determined to be an independent predictor of overall survival as higher expression of IRE1α in CRC patients showed lower survival rates (p = 0.0041). RNA interference and ectopic expression of IRE1α were applied to determine the molecular effects of IRE1α in CRC cells. The silencing of IRE1α inhibited the proliferation and blocked the invasion of CRC cells in vitro, while ectopic expression of IRE1α in turn promoted cell proliferation and invasion. IRE1α-XBP1 pathway regulated the mitosis of CRC cells through the directly binding of XBP1s to Cyclin D1 promoter to activate Cyclin D1 expression. Our results reveal that IRE1α-XBP1 pathway plays an important role in tumor progression and epithelial-to-mesenchymal transition (EMT), and IRE1α could be employed as a novel prognostic marker and a promising therapeutic target for CRC. - Highlights: • IRE1 was determined to be an independent predictor of overall survival in CRC patient. • IRE1-XBP1 pathway promoted CRC cell proliferation through regulating Cyclin D1 expression. • IRE1-XBP1 pathway played important role in EMT of CRC cells.

  4. Activation of IRE1α-XBP1 pathway induces cell proliferation and invasion in colorectal carcinoma

    International Nuclear Information System (INIS)

    Jin, Chun; Jin, Zhao; Chen, Nian-zhao; Lu, Min; Liu, Chang-bao; Hu, Wan-Le; Zheng, Chen-guo

    2016-01-01

    Cell proliferation and tumor metastasis are considered as the main reasons for death in colorectal carcinoma (CRC). IRE1α-XBP1 pathway is the most conserved UPR pathways, which are activated during ER stress caused by the accumulation of unfolded or misfolded protein in the lumen of ER. Here, we demonstrated the critical role of IRE1α-XBP1 pathway and underlying molecular mechanism in cell proliferation and tumor metastasis in CRC. By the use of tissue microarray analysis of samples from 119 patients with CRC, IRE1α was determined to be an independent predictor of overall survival as higher expression of IRE1α in CRC patients showed lower survival rates (p = 0.0041). RNA interference and ectopic expression of IRE1α were applied to determine the molecular effects of IRE1α in CRC cells. The silencing of IRE1α inhibited the proliferation and blocked the invasion of CRC cells in vitro, while ectopic expression of IRE1α in turn promoted cell proliferation and invasion. IRE1α-XBP1 pathway regulated the mitosis of CRC cells through the directly binding of XBP1s to Cyclin D1 promoter to activate Cyclin D1 expression. Our results reveal that IRE1α-XBP1 pathway plays an important role in tumor progression and epithelial-to-mesenchymal transition (EMT), and IRE1α could be employed as a novel prognostic marker and a promising therapeutic target for CRC. - Highlights: • IRE1 was determined to be an independent predictor of overall survival in CRC patient. • IRE1-XBP1 pathway promoted CRC cell proliferation through regulating Cyclin D1 expression. • IRE1-XBP1 pathway played important role in EMT of CRC cells.

  5. PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1

    Science.gov (United States)

    Gao, Guozhen; Dhar, Surbhi

    2017-01-01

    Abstract The type II arginine methyltransferase PRMT5 is responsible for the symmetric dimethylation of histone to generate the H3R8me2s and H4R3me2s marks, which correlate with the repression of transcription. However, the protein level of a number of genes (MEP50, CCND1, MYC, HIF1a, MTIF and CDKN1B) are reported to be downregulated by the loss of PRMT5, while their mRNA levels remain unchanged, which is counterintuitive for PRMT5's proposed role as a transcription repressor. We noticed that the majority of the genes regulated by PRMT5, at the posttranscriptional level, express mRNA containing an internal ribosome entry site (IRES). Using an IRES-dependent reporter system, we established that PRMT5 facilitates the translation of a subset of IRES-containing genes. The heterogeneous nuclear ribonucleoprotein, hnRNP A1, is an IRES transacting factor (ITAF) that regulates the IRES-dependent translation of Cyclin D1 and c-Myc. We showed that hnRNP A1 is methylated by PRMT5 on two residues, R218 and R225, and that this methylation facilitates the interaction of hnRNP A1 with IRES RNA to promote IRES-dependent translation. This study defines a new role for PRMT5 regulation of cellular protein levels, which goes beyond the known functions of PRMT5 as a transcription and splicing regulator. PMID:28115626

  6. Dissection of Ire1 functions reveals stress response mechanisms uniquely evolved in Candida glabrata.

    Directory of Open Access Journals (Sweden)

    Taiga Miyazaki

    2013-01-01

    Full Text Available Proper protein folding in the endoplasmic reticulum (ER is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR, is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata.

  7. Physiological roles of Regulated Ire1 Dependent Decay

    Directory of Open Access Journals (Sweden)

    Dina S. Coelho

    2014-04-01

    Full Text Available Ire1 is an important transducer of the Unfolded Protein Response (UPR that is activated by the accumulation of misfolded proteins in the Endoplamic Reticulum (ER stress. Activated Ire1 mediates the splicing of an intron from the mRNA of Xbp1, causing a frame-shift during translation and introducing a new carboxyl domain in the Xbp1 protein, which only then becomes a fully functional transcription factor. Studies using cell culture systems demonstrated that Ire1 also promotes the degradation of mRNAs encoding mostly ER-targeted proteins, to reduce the load of incoming ER client proteins during ER stress. This process was called RIDD (regulated Ire1-dependent decay, but its physiological significance remained poorly characterized beyond cell culture systems. Here we review several recent studies that have highlighted the physiological roles of RIDD in specific biological paradigms, such as photoreceptor differentiation in Drosophila or mammalian liver and endocrine pancreas function. These studies demonstrate the importance of RIDD in tissues undergoing intense secretory function and highlight the physiologic role of RIDD during UPR activation in cells and organisms.

  8. Ribosome stalling regulates IRES-mediated translation in eukaryotes, a parallel to prokaryotic attenuation

    NARCIS (Netherlands)

    Fernandez, James; Yaman, Ibrahim; Huang, Charles; Liu, Haiyan; Lopez, Alex B.; Komar, Anton A.; Caprara, Mark G.; Merrick, William C.; Snider, Martin D.; Kaufman, Randal J.; Lamers, Wouter H.; Hatzoglou, Maria

    2005-01-01

    It was previously shown that the mRNA for the cat-1 Arg/Lys transporter is translated from an internal ribosome entry site (IRES) that is regulated by cellular stress. Amino acid starvation stimulated cat-1 translation via a mechanism that requires translation of an ORF in the mRNA leader and

  9. HCV IRES-mediated core expression in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ye Zhao

    Full Text Available The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.

  10. The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells.

    Directory of Open Access Journals (Sweden)

    Justin R Hassler

    2015-10-01

    Full Text Available Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s. Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α to initiate X-box-binding protein 1 (Xbp1 mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq, we show that unconventional Xbp1 mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated Ire1α deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon Ire1α deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP, SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, Ire1α deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure.

  11. IRESPred: Web Server for Prediction of Cellular and Viral Internal Ribosome Entry Site (IRES)

    Science.gov (United States)

    Kolekar, Pandurang; Pataskar, Abhijeet; Kulkarni-Kale, Urmila; Pal, Jayanta; Kulkarni, Abhijeet

    2016-01-01

    Cellular mRNAs are predominantly translated in a cap-dependent manner. However, some viral and a subset of cellular mRNAs initiate their translation in a cap-independent manner. This requires presence of a structured RNA element, known as, Internal Ribosome Entry Site (IRES) in their 5′ untranslated regions (UTRs). Experimental demonstration of IRES in UTR remains a challenging task. Computational prediction of IRES merely based on sequence and structure conservation is also difficult, particularly for cellular IRES. A web server, IRESPred is developed for prediction of both viral and cellular IRES using Support Vector Machine (SVM). The predictive model was built using 35 features that are based on sequence and structural properties of UTRs and the probabilities of interactions between UTR and small subunit ribosomal proteins (SSRPs). The model was found to have 75.51% accuracy, 75.75% sensitivity, 75.25% specificity, 75.75% precision and Matthews Correlation Coefficient (MCC) of 0.51 in blind testing. IRESPred was found to perform better than the only available viral IRES prediction server, VIPS. The IRESPred server is freely available at http://bioinfo.net.in/IRESPred/. PMID:27264539

  12. Xbp1-Independent Ire1 Signaling Is Required for Photoreceptor Differentiation and Rhabdomere Morphogenesis in Drosophila

    Directory of Open Access Journals (Sweden)

    Dina S. Coelho

    2013-11-01

    Full Text Available The unfolded protein response (UPR is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Ire1 signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. Here, we show that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD, including for the fatty acid transport protein (fatp. Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. Our results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm.

  13. IRES-mediated translation of foot-and-mouth disease virus (FMDV) in cultured cells derived from FMDV-susceptible and -insusceptible animals.

    Science.gov (United States)

    Kanda, Takehiro; Ozawa, Makoto; Tsukiyama-Kohara, Kyoko

    2016-03-31

    Foot-and-mouth disease virus (FMDV) possess a positive sense, single stranded RNA genome. Internal ribosomal entry site (IRES) element exists within its 5' untranslated region (5'UTR) of the viral RNA. Translation of the viral RNA is initiated by internal entry of the 40S ribosome within the IRES element. This process is facilitated by cellular factors known as IRES trans-acting factors (ITAFs). Foot-and-mouth disease (FMD) is host-restricted disease for cloven-hoofed animals such as cattle and pigs, but the factors determining the host range have not been identified yet. Although, ITAFs are known to promote IRES-mediated translation, these findings were confirmed only in cells derived from FMDV-insusceptible animals so far. We evaluated and compared the IRES-mediated translation activities among cell lines derived from four different animal species using bicistronic luciferase reporter plasmid, which possesses an FMDV-IRES element between Renilla and Firefly luciferase genes. Furthermore, we analyzed the effect of the cellular factors on IRES-mediated translation by silencing the cellular factors using siRNA in both FMDV-susceptible and -insusceptible animal cells. Our data indicated that IRES-mediated translational activity was not linked to FMDV host range. ITAF45 promoted IRES-mediated translation in all cell lines, and the effects of poly-pyrimidine tract binding protein (PTB) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were observed only in FMDV-susceptible cells. Thus, PTB and 4E-BP1 may influence the host range of FMDV. IRES-mediated translation activity of FMDV was not predictive of its host range. ITAF45 promoted IRES-mediated translation in all cells, and the effects of PTB and 4E-BP1 were observed only in FMDV-susceptible cells.

  14. Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution.

    Science.gov (United States)

    Quade, Nick; Boehringer, Daniel; Leibundgut, Marc; van den Heuvel, Joop; Ban, Nenad

    2015-07-08

    Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.

  15. IRES-dependent translational control during virus-induced endoplasmic reticulum stress and apoptosis

    Directory of Open Access Journals (Sweden)

    Paul eHanson

    2012-03-01

    Full Text Available Many virus infections and stresses can induce endoplasmic reticulum (ER stress response, a host self defense mechanism against viral invasion and stress. During this event, viral and cellular gene expression is actively regulated and often encounters a switching of the translation initiation from cap-dependent to IRES (internal ribosome entry sites-dependent. This switching is largely dependent on the mRNA structure of the 5’untranslated region (5’UTR and on the particular stress stimuli. Picornviruses and some other viruses contain an IRES within their 5’UTR of viral genome and employ an IRES-driven mechanism for translation initiation. Recently, a growing number of cellular genes involved in growth control, cell cycle progression and apoptosis were also found to contain one or more IRES within their long highly structured 5’UTRs. These genes initiate translation usually by a cap-dependent mechanism under normal physiological conditions; however, in certain environments, such as infection, starvation and heat shock they shift translation initiation to an IRES-dependent modality. Although the molecular mechanism is not entirely understood, a number of studies have revealed that several cellular biochemical processes are responsible for the switching of translation initiation to IRES-dependent. These include the cleavage of translation initiation factors by viral and/or host proteases, phosphorylation (inactivation of host factors for translation initiation, over-production of homologous proteins of cap-binding protein eIF4E, suppression of cap-binding protein eIF4E expression by specific microRNA, activation of enzymes for mRNA decapping, as well as others. Here, we summarize the recent advances in our understanding of the molecular mechanisms for the switching of translation initiation, particularly for the proteins involved in cell survival and apoptosis in the ER stress pathways during viral infections.

  16. IRSS: a web-based tool for automatic layout and analysis of IRES secondary structure prediction and searching system in silico

    Directory of Open Access Journals (Sweden)

    Hong Jun-Jie

    2009-05-01

    Full Text Available Abstract Background Internal ribosomal entry sites (IRESs provide alternative, cap-independent translation initiation sites in eukaryotic cells. IRES elements are important factors in viral genomes and are also useful tools for bi-cistronic expression vectors. Most existing RNA structure prediction programs are unable to deal with IRES elements. Results We designed an IRES search system, named IRSS, to obtain better results for IRES prediction. RNA secondary structure prediction and comparison software programs were implemented to construct our two-stage strategy for the IRSS. Two software programs formed the backbone of IRSS: the RNAL fold program, used to predict local RNA secondary structures by minimum free energy method; and the RNA Align program, used to compare predicted structures. After complete viral genome database search, the IRSS have low error rate and up to 72.3% sensitivity in appropriated parameters. Conclusion IRSS is freely available at this website http://140.135.61.9/ires/. In addition, all source codes, precompiled binaries, examples and documentations are downloadable for local execution. This new search approach for IRES elements will provide a useful research tool on IRES related studies.

  17. Parallel Auxiliary Space AMG Solver for $H(div)$ Problems

    Energy Technology Data Exchange (ETDEWEB)

    Kolev, Tzanio V. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2012-12-18

    We present a family of scalable preconditioners for matrices arising in the discretization of $H(div)$ problems using the lowest order Raviart--Thomas finite elements. Our approach belongs to the class of “auxiliary space''--based methods and requires only the finite element stiffness matrix plus some minimal additional discretization information about the topology and orientation of mesh entities. Also, we provide a detailed algebraic description of the theory, parallel implementation, and different variants of this parallel auxiliary space divergence solver (ADS) and discuss its relations to the Hiptmair--Xu (HX) auxiliary space decomposition of $H(div)$ [SIAM J. Numer. Anal., 45 (2007), pp. 2483--2509] and to the auxiliary space Maxwell solver AMS [J. Comput. Math., 27 (2009), pp. 604--623]. Finally, an extensive set of numerical experiments demonstrates the robustness and scalability of our implementation on large-scale $H(div)$ problems with large jumps in the material coefficients.

  18. Phyto-gegographycal analysis of Divčibare

    Directory of Open Access Journals (Sweden)

    Popović Ivana

    2005-01-01

    Full Text Available The aim of phytogeographical analysis is to determine the plant species origin, genesis and migrations. The study results contribute to the knowledge of phytogeographical classification of the particular regions. Different chorions, by their effects, determine the flora structure and composition. The aim of the floristic research of Divčibare was to identify the diversity of flora in this region, i.e., in a wider sense, the diversity of flora in Serbia. Phytogeographical analysis was based on floristic research. The identified plant species were classified into the corresponding floral elements, area groups and area types. The data on diversity and the degree of representation in the flora of Divčibare were obtained based on the percentage of each area type. It was concluded that the flora of Divčibare has a transitional, Central European-Mediterranean-Pontic character.

  19. Characterization of the functional role of nucleotides within the URE2 IRES element and the requirements for eIF2A-mediated repression.

    Science.gov (United States)

    Reineke, Lucas C; Merrick, William C

    2009-12-01

    Cap-independent initiation of translation is thought to promote protein synthesis on some mRNAs during times when cap-dependent initiation is down-regulated. However, the mechanism of cap-independent initiation is poorly understood. We have previously reported the secondary structure within the yeast minimal URE2 IRES element. In this study, we sought to investigate the mechanism of internal initiation in yeast by assessing the functional role of nucleotides within the minimal URE2 IRES element, and delineating the cis-sequences that modulate levels of internal initiation using a monocistronic reporter vector. Furthermore, we compared the eIF2A sensitivity of the URE2 IRES element with some of the invasive growth IRES elements using DeltaeIF2A yeast. We found that the stability of the stem-loop structure within the minimal URE2 IRES element is not a critical determinant of optimal IRES activity, and the downstream sequences that modulate URE2 IRES-mediated translation can be defined to discrete regions within the URE2 coding region. Repression of internal initiation on the URE2 minimal IRES element by eIF2A is not dependent on the stability of the secondary structure within the URE2 IRES element. Our data also indicate that eIF2A-mediated repression is not specific to the URE2 IRES element, as both the GIC1 and PAB1 IRES elements are repressed by eIF2A. These data provide valuable insights into the mRNA requirements for internal initiation in yeast, and insights into the mechanism of eIF2A-mediated suppression.

  20. The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

    Science.gov (United States)

    Pini, Francesco; Frage, Benjamin; Ferri, Lorenzo; De Nisco, Nicole J.; Mohapatra, Saswat S.; Taddei, Lucilla; Fioravanti, Antonella; Dewitte, Frederique; Galardini, Marco; Brilli, Matteo; Villeret, Vincent; Bazzicalupo, Marco; Mengoni, Alessio; Walker, Graham C.; Becker, Anke; Biondi, Emanuele G.

    2013-01-01

    SUMMARY Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis. PMID:23909720

  1. Characterization of Hepatitis C Virus IRES Quasispecies – From the Individual to the Pool

    Directory of Open Access Journals (Sweden)

    Václav Vopálenský

    2018-04-01

    Full Text Available Hepatitis C virus (HCV is a single-stranded positive-sense RNA virus from the genus Hepacivirus. The viral genomic +RNA is 9.6 kb long and contains highly structured 5′ and 3′ untranslated regions (UTRs and codes for a single large polyprotein, which is co- and post-translationally processed by viral and cellular proteases into at least 11 different polypeptides. Most of the 5′ UTR and an initial part of the polyprotein gene are occupied by an internal ribosome entry site (IRES, which mediates cap-independent translation of the viral proteins and allows the virus to overcome cellular antiviral defense based on the overall reduction of the cap-dependent translation initiation. We reconsidered published results concerning a search for possible correlation between patient response to interferon-based antiviral therapy and accumulation of nucleotide changes within the HCV IRES. However, we were unable to identify any such correlation. Rather than searching for individual mutations, we suggest to focus on determination of individual and collective activities of the HCV IRESs found in patient specimens. We developed a combined, fast, and undemanding approach based on high-throughput cloning of the HCV IRES species to a bicistronic plasmid followed by determination of the HCV IRES activity by flow cytometry. This approach can be adjusted for measurement of the individual HCV IRES activity and for estimation of the aggregate ability of the whole HCV population present in the specimen to synthesize viral proteins. To detect nucleotide variations in the individual IRESs, we used denaturing gradient gel electrophoresis (DGGE analysis that greatly improved identification and classification of HCV IRES variants in the sample. We suggest that determination of the collective activity of the majority of HCV IRES variants present in one patient specimen in a given time represents possible functional relations among variant sequences within the complex

  2. Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved

    International Nuclear Information System (INIS)

    Thompson, Sunnie R.; Sarnow, Peter

    2003-01-01

    Human enterovirus 71 (EV71) is a member of the Enterovirus genus of the Picornaviridae family. Other members of this family utilize an unusual mechanism of translation initiation whereby ribosomes are recruited internally to the viral RNA by an internal ribosome site (IRES) located in their 5' noncoding regions (5' NCR). Using dicistronic reporter constructs, we demonstrate that the 5' NCRs of the 7423/MS/87 and BrCr strains of EV71 function as an IRES both in extracts and in cultured cells. Preincubation of translation extracts with purified coxsackievirus 2A protease cleaved elF4G, a component of the cap binding complex, resulting in a significant decrease in translation of capped mRNAs. In contrast, the translational efficiency of the EV71 IRES was enhanced under this condition, demonstrating that the EV71 IRES functions similar to other enterovirus IRES elements when components of the cap binding protein complex are cleaved. Finally, insertion of an upstream, out-of-frame start codon in the 5' NCR of the EV71 genome inhibited IRES activity, suggesting that EV71 can be classified as a type I IRES, in which ribosomes first bind upstream of the initiation codon and then scan the mRNA until an appropriate downstream AUG start codon is encountered and protein synthesis commences

  3. Targeting the CACNA1A IRES as a Treatment for Spinocerebellar Ataxia Type 6.

    Science.gov (United States)

    Pastor, Parviz Daniel Hejazi; Du, Xiaofei; Fazal, Sarah; Davies, Andre N; Gomez, Christopher M

    2018-02-01

    We have discovered that the P/Q-type voltage-gated Ca 2+ channel (VGCC) gene, CACNA1A, encodes both the α1A (Cav2.1) subunit and a newly recognized transcription factor, α1ACT, by means of a novel internal ribosomal entry site (IRES) within the α1A C-terminal coding region. α1ACT, when mutated with an expansion of the polyglutamine tract in the C-terminus, gives rise to spinocerebellar ataxia type 6 (SCA6). Because silencing of the entire CACNA1A gene would result in the loss of the essential Cav2.1 channel, the IRES controlling α1ACT expression is an excellent target for selective silencing of α1ACT as a therapeutic intervention for SCA6. We performed a high-throughput screen of FDA-approved small molecules using a dual luciferase reporter system and identified ten hits able to selectively inhibit the IRES. We identified four main candidates that showed selective suppression of α1ACT relative to α1A in HEK cells expressing a native CACNA1A vector. We previously pursued another avenue of molecular intervention through miRNA silencing. We studied three human miRNAs (miRNA-711, -3191-5p, -4786) that would potentially bind to sequences within the CACNA1A IRES region, based on an miRNA prediction program. Only miRNA-3191-5p was found to selectively inhibit the translation of α1ACT in cells. We developed a hyperacute model of SCA6 in mice by injecting a pathogenic form of the IRES-mediated α1ACT (AAV9-α1ACTQ33). Finally, we tested the effectiveness of the miRNA therapy by co-expressing either control miRNA or miRNA-3191-5p and found that miRNA-3191-5p decreased the levels of α1ACTQ33 and prevented the hyperacute disease in mice. These studies provide the proof of principle that a therapy directed at selectively preventing α1ACT expression could be used to treat SCA6.

  4. Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line.

    Science.gov (United States)

    Kawaguchi, Daichi; Sahara, Setsuko; Zembrzycki, Andreas; O'Leary, Dennis D M

    2016-04-01

    Foxg1 expression is highly restricted to the telencephalon and other head structures in the early embryo. This expression pattern has been exploited to generate conditional knockout mice, based on a widely used Foxg1-Cre knock-in line (Foxg1(tm1(cre)Skm)), in which the Foxg1 coding region was replaced by the Cre gene. The utility of this line, however, is severely hampered for two reasons: (1) Foxg1-Cre mice display ectopic and unpredictable Cre activity, and (2) Foxg1 haploinsufficiency can produce neurodevelopmental phenotypes. To overcome these issues, we have generated a new Foxg1-IRES-Cre knock-in mouse line, in which an IRES-Cre cassette was inserted in the 3'UTR of Foxg1 locus, thus preserving the endogenous Foxg1 coding region and un-translated gene regulatory sequences in the 3'UTR, including recently discovered microRNA target sites. We further demonstrate that the new Foxg1-IRES-Cre line displays consistent Cre activity patterns that recapitulated the endogenous Foxg1 expression at embryonic and postnatal stages without causing defects in cortical development. We conclude that the new Foxg1-IRES-Cre mouse line is a unique and advanced tool for studying genes involved in the development of the telencephalon and other Foxg1-expressing regions starting from early embryonic stages. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Effect of varying temperature on growth, morphology and soluble protein content of div I and div II mutant strains of bacillus sub tills

    International Nuclear Information System (INIS)

    Ahmed, A.; Sabri, A.N.

    2004-01-01

    In B.subtilis, cell division is controlled by div-genes which have been mapped on its circular chromosome. In the present work, div-mutant strains 1A316(div II), 1A317 and 1A318 (div I) were studied. These strains exhibited temperature sensitive cell division mutations. Colony morphology, cell morphology, staining behavior, growth rate and protein content of PY79 (wild type) and div-mutant strains (1A316, 1A317, 1A318) was studied at different temperatures ( 25 deg. Centi grade and 42 deg. with varying incubation periods(4, 16, 24, 48, 72,96 hrs). div-mutants differ from wild type (PY79) in colony morphology. Colony margin in PY79 was entire while in the div strains it is undulate. Staining behavior of cells as well as cell morphology i.e., cell size, cell types, formation of filaments/minicells were affected by high temperature. At higher temperature (42 deg. Centi grade), div-mutants undergo more severe lysis and degeneration as compare to wild type (PY79). Defective spores were produced by div-mutants at 25 deg. Centi grade and 42 deg. Centi grade. Tetrazolium overlay test was performed at 37 deg. Centi grade and 42 deg. Centi grade to check the spore germination ability of wild type and div-mutants. In 1A318, defective spores were produced at 37 deg. Centi grade, div-mutant was checked after 24 and 96 hrs at different temperatures (25, 37 and 42 deg. Centi grade). At all temperatures protein content were more in PY79 as compare to div-mutants. Also at 25 and 42 deg. Centi grade, protein content was more as compare to 37 deg. Centi grade. Protein contents was reduced at sporulation stages. Thus cell division mutations affect cell morphology, sporulation and germination processes in B.subtilis and thus are multifaceted mutations. (author)

  6. IRE1: ER stress sensor and cell fate executor.

    Science.gov (United States)

    Chen, Yani; Brandizzi, Federica

    2013-11-01

    Cells operate a signaling network termed the unfolded protein response (UPR) to monitor protein-folding capacity in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) is an ER transmembrane sensor that activates the UPR to maintain the ER and cellular function. Although mammalian IRE1 promotes cell survival, it can initiate apoptosis via decay of antiapoptotic miRNAs. Convergent and divergent IRE1 characteristics between plants and animals underscore its significance in cellular homeostasis. This review provides an updated scenario of the IRE1 signaling model, discusses emerging IRE1 sensing mechanisms, compares IRE1 features among species, and outlines exciting future directions in UPR research. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Alphavirus replicon approach to promoterless analysis of IRES elements.

    Science.gov (United States)

    Kamrud, K I; Custer, M; Dudek, J M; Owens, G; Alterson, K D; Lee, J S; Groebner, J L; Smith, J F

    2007-04-10

    Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

  8. IRE1α links Nck1 deficiency to attenuated PTP1B expression in HepG2 cells.

    Science.gov (United States)

    Li, Hui; Li, Bing; Larose, Louise

    2017-08-01

    PTP1B, a prototype of the non-receptor subfamily of the protein tyrosine phosphatase superfamily, plays a key role in regulating intracellular signaling from various receptor and non-receptor protein tyrosine kinases. Previously, we reported that silencing Nck1 in human hepatocellular carcinoma HepG2 cells enhances basal and growth factor-induced activation of the PI3K-Akt pathway through attenuating PTP1B expression. However, the underlying mechanism by which Nck1 depletion represses PTP1B expression remains unclear. In this study, we found that silencing Nck1 attenuates PTP1B expression in HepG2 cells through down-regulation of IRE1α. Indeed, we show that silencing Nck1 in HepG2 cells leads to decreased IRE1α expression and signaling. Accordingly, IRE1α depletion using siRNA in HepG2 cells enhances PI3K-dependent basal and growth factor-induced Akt activation, reproducing the effects of silencing Nck1 on activation of this pathway. In addition, depletion of IRE1α also leads to reduced PTP1B expression, which was rescued by ectopic expression of IRE1α in Nck1-depleted cells. Mechanistically, we found that silencing either Nck1 or IRE1α in HepG2 cells decreases PTP1B mRNA levels and stability. However, despite miR-122 levels, a miRNA targeting PTP1B 3' UTR and inducing PTP1B mRNA degradation in HepG2 cells, are increased in both Nck1- and IRE1α-depleted HepG2 cells, a miR-122 antagomir did not rescue PTP1B expression in these cells. Overall, this study highlights an important role for Nck1 in fine-tuning IRE1α expression and signaling that regulate PTP1B expression and subsequent activation of the PI3K-Akt pathway in HepG2 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. The effects of irreversible electroporation (IRE on nerves.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available BACKGROUND: If a critical nerve is circumferentially involved with tumor, radical surgery intended to cure the cancer must sacrifice the nerve. Loss of critical nerves may lead to serious consequences. In spite of the impressive technical advancements in nerve reconstruction, complete recovery and normalization of nerve function is difficult to achieve. Though irreversible electroporation (IRE might be a promising choice to treat tumors near or involved critical nerve, the pathophysiology of the nerve after IRE treatment has not be clearly defined. METHODS: We applied IRE directly to a rat sciatic nerve to study the long term effects of IRE on the nerve. A sequence of 10 square pulses of 3800 V/cm, each 100 µs long was applied directly to rat sciatic nerves. In each animal of group I (IRE the procedure was applied to produce a treated length of about 10 mm. In each animal of group II (Control the electrodes were only applied directly on the sciatic nerve for the same time. Electrophysiological, histological, and functional studies were performed on immediately after and 3 days, 1 week, 3, 5, 7 and 10 weeks following surgery. FINDINGS: Electrophysiological, histological, and functional results show the nerve treated with IRE can attain full recovery after 7 weeks. CONCLUSION: This finding is indicative of the preservation of nerve involving malignant tumors with respect to the application of IRE pulses to ablate tumors completely. In summary, IRE may be a promising treatment tool for any tumor involving nerves.

  10. The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

    Science.gov (United States)

    Jaffrey, S R; Haile, D J; Klausner, R D; Harford, J B

    1993-09-25

    To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.

  11. Analysis of biological spectrum of Divčibare flora

    Directory of Open Access Journals (Sweden)

    Popović Ivana

    2006-01-01

    Full Text Available One of the essential analyses which is performed during the floristic study of a region is the analysis of the biological spectrum. The analysis of the biological spectrum of the flora includes the determination of the type of life form for each taxon described in the flora of the study region. If it is considered that life form is a specific structural-functional response to the environmental effects and the result of the adaptation during the species evolution, it is clear that the basic characteristics of the site are more or less reflected in any life form. This fact is confirmed by the analysis of the biological spectrum of Divčibare flora. The study results are in correlation with the results of the analysis of the biological spectrum of the flora of Serbia and the Balkan Peninsula.

  12. Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor.

    Science.gov (United States)

    Son, S M; Byun, J; Roh, S-E; Kim, S J; Mook-Jung, I

    2014-04-17

    The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca(2+). Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca(2+) homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca(2+) dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca(2+) concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca(2+) through the InsP3 receptor (InsP3R). The Ca(2+) efflux in IRE1α-deficient cells correlates with dissociation of the Ca(2+)-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α-TRAF2-ASK1 complex. The increased cytosolic concentration of Ca(2+) induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca(2+) dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca(2+) influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca(2+) homeostasis and cell survival during ER stress and reveal a previously unknown Ca(2+)-mediated cell death signaling between the IRE1α-InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.

  13. High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor

    International Nuclear Information System (INIS)

    Auf, Gregor; Vajkoczy, Peter; Seno, Masaharu; Bikfalvi, Andreas; Minchenko, Dmitri; Minchenko, Oleksandr; Moenner, Michel; Jabouille, Arnaud; Delugin, Maylis; Guérit, Sylvaine; Pineau, Raphael; North, Sophie; Platonova, Natalia; Maitre, Marlène; Favereaux, Alexandre

    2013-01-01

    Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma. Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown. EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α. EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the

  14. New small molecule inhibitors of UPR activation demonstrate that PERK, but not IRE1α signaling is essential for promoting adaptation and survival to hypoxia

    International Nuclear Information System (INIS)

    Cojocari, Dan; Vellanki, Ravi N.; Sit, Brandon; Uehling, David; Koritzinsky, Marianne; Wouters, Bradly G.

    2013-01-01

    Background and purpose: The unfolded protein response (UPR) is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling arms. Here we explore the potential of targeting two of these arms with new potent small-molecule inhibitors designed against IRE1α and PERK. Methods: We utilized shRNAs and small-molecule inhibitors of IRE1α (4μ8c) and PERK (GSK-compound 39). XBP1 splicing and DNAJB9 mRNA was measured by qPCR and was used to monitor IRE1α activity. PERK activity was monitored by immunoblotting eIF2α phosphorylation and qPCR of DDIT3 mRNA. Hypoxia tolerance was measured using proliferation and clonogenic cell survival assays of cells exposed to mild or severe hypoxia in the presence of the inhibitors. Results: Using knockdown experiments we show that PERK is essential for survival of KP4 cells while knockdown of IRE1α dramatically decreases the proliferation and survival of HCT116 during hypoxia. Further, we show that in response to both hypoxia and other ER stress-inducing agents both 4μ8c and the PERK inhibitor are selective and potent inhibitors of IRE1α and PERK activation, respectively. However, despite potent inhibition of IRE1α activation, 4μ8c had no effect on cell proliferation or clonogenic survival of cells exposed to hypoxia. This was in contrast to the inactivation of PERK signaling with the PERK inhibitor, which reduced tolerance to hypoxia and other ER stress inducing agents. Conclusions: Our results demonstrate that IRE1α but not its splicing activity is important for hypoxic cell survival. The PERK signaling arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress and should be the focus of future therapeutic efforts

  15. Endoplasmic reticulum stress and IRE-1 signaling cause apoptosis in colon cancer cells in response to andrographolide treatment.

    Science.gov (United States)

    Banerjee, Aditi; Ahmed, Hafiz; Yang, Peixin; Czinn, Steven J; Blanchard, Thomas G

    2016-07-05

    The plant metabolite andrographolide induces cell cycle arrest and apoptosis in cancer cells. The mechanism(s) by which andrographolide induces apoptosis however, have not been elucidated. The present study was performed to determine the molecular events that promote apoptosis in andrographolide treated cells using T84, HCT116 and COLO 205 colon cancer cell lines. Andrographolide was determined to limit colony formation and Ki67 expression, alter nuclear morphology, increase cytoplasmic histone-associated-DNA-fragments, and increase cleaved caspase-3 levels. Andrographolide also induced significantly higher expression of endoplasmic reticulum (ER) stress proteins GRP-78 and IRE-1 by 48 h but not PERK or ATF6. Apoptosis signaling molecules BAX, spliced XBP-1 and CHOP were also significantly increased. Moreover, chemical inhibition of ER stress or IRE-1 depletion with siRNA in andrographolide treated cells significantly limited expression of IRE-1 and CHOP as determined by immunofluorescence staining, real time PCR, or immunobloting. This was accompanied by a decreased BAX/Bcl-2 ratio. Andrographolide significantly promotes cancer cell death compared to normal cells. These data demonstrate that andrographolide associated ER stress contributes to apoptosis through the activation of a pro-apoptotic GRP-78/IRE-1/XBP-1/CHOP signaling pathway.

  16. Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains.

    Science.gov (United States)

    Hsu, Eric J; Zhu, Wandi; Schubert, Angela R; Voelker, Taylor; Varga, Zoltan; Silva, Jonathan R

    2017-03-06

    Functional eukaryotic voltage-gated Na + (Na V ) channels comprise four domains (DI-DIV), each containing six membrane-spanning segments (S1-S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1-S4), which together form a voltage-sensing domain (VSD). A critical Na V channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair Na V channel inactivation. These locations include the DIII-DIV linker, the DIII S4-S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII-DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery. © 2017 Hsu et al.

  17. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

    Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha\\/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  18. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

    Directory of Open Access Journals (Sweden)

    Sanjeev Gupta

    2010-07-01

    Full Text Available Endoplasmic reticulum (ER stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR. Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  19. RITA enhances irradiation-induced apoptosis in p53-defective cervical cancer cells via upregulation of IRE1α/XBP1 signaling.

    Science.gov (United States)

    Zhu, Hong; Abulimiti, Muyasha; Liu, Huan; Su, Xiang-Jiang; Liu, Cai-Hong; Pei, Hai-Ping

    2015-09-01

    Radiation therapy is the most widely used treatment for patients with cervical cancer. Recent studies have shown that endoplasmic reticulum (ER) stress induces apoptosis and sensitizes tumor cells to radiotherapy, which reportedly induces ER stress in cells. Classical key tumor suppressor p53 is involved in the response to a variety of cellular stresses, including those incurred by ionizing irradiation. A recent study demonstrated that small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) increased the radiosensitivity of tumor cells expressing mutant p53 (mtp53). In the present study, we explored the effects and the underlying mechanisms of RITA in regards to the radiosensitivity and ER stress in mtp53-expressing human cervix cancer cells. Treatment with 1 µM of RITA for 24 h before irradiation markedly decreased survival and increased apoptosis in C-33A and HT-3 cells; the effects were not significantly altered by knockdown of p53. In the irradiated C-33A and HT-3 cells, RITA significantly increased the expression of IRE1α, the spliced XBP1 mRNA level, as well as apoptosis; the effects were abolished by knockdown of IRE1α. Transcriptional pulse-chase assays revealed that RITA significantly increased the stability of IRE1α mRNA in the irradiated C-33A and HT-3 cells. In contrast, the same RITA treatment did not show any significant effect on sham-irradiated cells. In conclusion, the present study provides initial evidence that RITA upregulates the expression level of IRE1α by increasing the stability of IRE1α mRNA in irradiated mtp53-expressing cervical cancer cells; the effect leads to enhanced IRE1α/XBP1 ER stress signaling and increased apoptosis in the cells. The present study offers novel insight into the pharmacological potential of RITA in the radiotherapy for cervical cancer.

  20. SecA is required for membrane targeting of the cell division protein DivIVA in vivo

    Directory of Open Access Journals (Sweden)

    Sven eHalbedel

    2014-02-01

    Full Text Available The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localises to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localisation of DivIVA in these mutants. Surprisingly, DivIVA was delocalised, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localisation depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localisation is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

  1. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  2. Ire1 Has Distinct Catalytic Mechanisms for XBP1/HAC1 Splicing and RIDD

    Directory of Open Access Journals (Sweden)

    Arvin B. Tam

    2014-11-01

    Full Text Available An evolutionarily conserved unfolded protein response (UPR component, IRE1, cleaves XBP1/HAC1 introns in order to generate spliced mRNAs that are translated into potent transcription factors. IRE1 also cleaves endoplasmic-reticulum-associated RNAs leading to their decay, an activity termed regulated IRE1-dependent decay (RIDD; however, the mechanism by which IRE1 differentiates intron cleavage from RIDD is not well understood. Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase and mutations at oligomerization interfaces. Using an IRE1 RNase inhibitor, STF-083010, selective inhibition of XBP1 splicing indicates that XBP1 promotes cell survival, whereas RIDD leads to cell death, revealing modulation of IRE1 activities as a drug-development strategy.

  3. Parallel Solver for H(div) Problems Using Hybridization and AMG

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Chak S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-01-15

    In this paper, a scalable parallel solver is proposed for H(div) problems discretized by arbitrary order finite elements on general unstructured meshes. The solver is based on hybridization and algebraic multigrid (AMG). Unlike some previously studied H(div) solvers, the hybridization solver does not require discrete curl and gradient operators as additional input from the user. Instead, only some element information is needed in the construction of the solver. The hybridization results in a H1-equivalent symmetric positive definite system, which is then rescaled and solved by AMG solvers designed for H1 problems. Weak and strong scaling of the method are examined through several numerical tests. Our numerical results show that the proposed solver provides a promising alternative to ADS, a state-of-the-art solver [12], for H(div) problems. In fact, it outperforms ADS for higher order elements.

  4. Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

    Directory of Open Access Journals (Sweden)

    Anne Dallas

    2013-01-01

    Full Text Available We previously identified short synthetic shRNAs (sshRNAs that target a conserved hepatitis C virus (HCV sequence within the internal ribosome entry site (IRES of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA, SG220 was formulated into lipid nanoparticles (LNP and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and 3H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% 1 day after injection of 2.5 mg/kg sshRNA with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.

  5. Promotion of Viral IRES-Mediated Translation Initiation under Mild Hypothermia.

    Directory of Open Access Journals (Sweden)

    Maria Licursi

    Full Text Available Internal ribosome entry site (IRES-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV, foot-and-mouth disease virus (FMDV, hepatitis C virus (HCV, human rhinovirus (HRV or poliovirus (PV. Under mild hypothermic conditions (30 and 35°C, we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB and upstream of N-Ras (unr, the IRES trans-acting factors (ITAFs of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent.

  6. Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation.

    Science.gov (United States)

    Soto Rifo, Ricardo; Ricci, Emiliano P; Décimo, Didier; Moncorgé, Olivier; Ohlmann, Théophile

    2007-01-01

    Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

  7. Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA.

    Directory of Open Access Journals (Sweden)

    Prahathees Eswaramoorthy

    2014-08-01

    Full Text Available Sporulation in the bacterium Bacillus subtilis is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σF, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σF. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σF is activated.

  8. Spectral mimetic least-squares method for div-curl systems

    NARCIS (Netherlands)

    Gerritsma, Marc; Palha, Artur; Lirkov, I.; Margenov, S.

    2018-01-01

    In this paper the spectral mimetic least-squares method is applied to a two-dimensional div-curl system. A test problem is solved on orthogonal and curvilinear meshes and both h- and p-convergence results are presented. The resulting solutions will be pointwise divergence-free for these test

  9. The IRP/IRE system in vivo: insights from mouse models

    Directory of Open Access Journals (Sweden)

    Nicole eWilkinson

    2014-07-01

    Full Text Available Iron regulatory proteins 1 and 2 (IRP1 and IRP2 post-transcriptionally control the expression of several mRNAs encoding proteins of iron, oxygen and energy metabolism. The mechanism involves their binding to iron responsive elements (IREs in the untranslated regions of target mRNAs, thereby controlling mRNA translation or stability. Whereas IRP2 functions solely as an RNA-binding protein, IRP1 operates as either an RNA-binding protein or a cytosolic aconitase. Early experiments in cultured cells established a crucial role of IRPs in regulation of cellular iron metabolism. More recently, studies in mouse models with global or localized Irp1 and/or Irp2 deficiencies uncovered new physiological functions of IRPs in the context of systemic iron homeostasis. Thus, IRP1 emerged as a key regulator of erythropoiesis and iron absorption by controlling hypoxia inducible factor 2α (HIF2α mRNA translation, while IRP2 appears to dominate the control of iron uptake and heme biosynthesis in erythroid progenitor cells by regulating the expression of transferrin receptor 1 (TfR1 and 5-aminolevulinic acid synthase 2 (ALAS2 mRNAs, respectively. Targeted disruption of either Irp1 or Irp2 in mice is associated with distinct phenotypic abnormalities. Thus, Irp1-/- mice develop polycythemia and pulmonary hypertension, while Irp2-/- mice present with microcytic anemia, iron overload in the intestine and the liver, and neurologic defects. Combined disruption of both Irp1 and Irp2 is incombatible with life and leads to early embryonic lethality. Mice with intestinal- or liver-specific disruption of both Irps are viable at birth but die later on due to malabsorption or liver failure, respectively. Adult mice lacking both Irps in the intestine exhibit a profound defect in dietary iron absorption due to a mucosal block that is caused by the de-repression of ferritin mRNA translation. Herein, we discuss the physiological function of the IRE/IRP regulatory system.

  10. Beam Simulations for IRE and Driver-Status and Strategy

    International Nuclear Information System (INIS)

    Friedman, A.; Grote, D.P.; Lee, E.P.; Sonnendrucker, E.

    2000-01-01

    The methods and codes employed in the U.S. Heavy Ion Fusion program to simulate the beams in an Integrated Research Experiments (IRE) facility and a fusion driver are presented in overview. A new family of models incorporating accelerating module impedance, multi-beam, and self-magnetic effects is described, and initial WARP3d particle simulations of beams using these models are presented. Finally, plans for streamlining the machine-design simulation sequence, and for simulating beam dynamics from the source to the target in a consistent and comprehensive manner, are described

  11. A weak Galerkin least-squares finite element method for div-curl systems

    Science.gov (United States)

    Li, Jichun; Ye, Xiu; Zhang, Shangyou

    2018-06-01

    In this paper, we introduce a weak Galerkin least-squares method for solving div-curl problem. This finite element method leads to a symmetric positive definite system and has the flexibility to work with general meshes such as hybrid mesh, polytopal mesh and mesh with hanging nodes. Error estimates of the finite element solution are derived. The numerical examples demonstrate the robustness and flexibility of the proposed method.

  12. IRE/F as a Cross-Curricular Collaborative Genre of Implicit Argumentation

    Science.gov (United States)

    Lawrence, Ann M.; Crespo, Sandra

    2016-01-01

    We contend that the classroom-discourse routine of IRE/F (teacher initiation, student response, teacher evaluation/follow-up) is a genre of argumentation that is both collaborative and implicit because teachers and students cooperate during IRE/F exchanges not only to make and mirror knowledge claims but also to suppress justification for those…

  13. Effect of CD34+ cord blood stem cell transfected by plasmid vector pIRES2-FL-IL-3 on the mice after irradiation

    International Nuclear Information System (INIS)

    Zhang Yong; Zhang Linsheng; Zhang Hongbing; Guo Chaohua; Tong Shiwu

    2008-01-01

    Objective: To observe the effect of CD34 cord blood stem cell transfected by plasmid vector plRES2-FL-IL-3 on the mouse after irradiation and to investigate its mechanisms. Methods: In the co-expressed group (12 mice), CD 34 + cord blood stem cells were transfected by plasmid vector pIRES2-FL-IL-3.5 x lO 5 cells were injected intravenously in the mouse. The hemogram changes in mice were detected 2, 4 and 6 weeks after radiation. At 6 weeks after irradiation, the expression of the CD 34 in spleen was detected by immumofluorescence method. The mRNA level and the activity of IL-3 and FL were detected by RT-PCR and Western blot. Other 3 groups were CD 34 + cell group (CD 34 group), pIRES2-IL-3 group(IL 3 group) and pIRES2-FL group(FL group), and there were 12 mice in each group. Results: The survival rate of CD 34 group, IL3 group and FL group at the 6th week were 25.0% (3/12), 50.0% (6/12) and 50.0% (6/12), respectively. It was 91.7% (11/12) in the co-expressed group, which was higher than those in the other groups. The expression of the CD 34 of spleen in the co-expressed group was higher than those of the other groups. The mRNA level and the activity of IL-3 and FL of spleen in the co-expressed group were higher than those in the other groups too. Conclusions: The CD 34 '+ cord blood stem cells transfected by plasmid vector pIRES2-FL-IL-3 have hemogenesis promotion effect on the mice after irradiation, which was related with the aggregation, proliferation of stem cells and the high expression of the interest proteins.. (authors)

  14. 42 CFR 423.2010 - When CMS, the IRE, or Part D plan sponsors may participate in an ALJ hearing.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false When CMS, the IRE, or Part D plan sponsors may... PRESCRIPTION DRUG BENEFIT Reopening, ALJ Hearings, MAC review, and Judicial Review § 423.2010 When CMS, the IRE... require, CMS, the IRE, and/or the Part D plan sponsor to participate in any proceedings before the ALJ...

  15. hNIS-IRES-eGFP Dual Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Jiantu Che

    2005-04-01

    Full Text Available The human and rodent sodium iodide symporters (NIS have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES-linked human NIS (hNIS-enhanced green fluorescent protein (eGFP hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4– to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.

  16. The UPR reduces glucose metabolism via IRE1 signaling.

    Science.gov (United States)

    van der Harg, Judith M; van Heest, Jessica C; Bangel, Fabian N; Patiwael, Sanne; van Weering, Jan R T; Scheper, Wiep

    2017-04-01

    Neurons are highly dependent on glucose. A disturbance in glucose homeostasis therefore poses a severe risk that is counteracted by activation of stress responses to limit damage and restore the energy balance. A major stress response that is activated under conditions of glucose deprivation is the unfolded protein response (UPR) that is aimed to restore proteostasis in the endoplasmic reticulum. The key signaling of the UPR involves the transient activation of a transcriptional program and an overall reduction of protein synthesis. Since the UPR is strategically positioned to sense and integrate metabolic stress signals, it is likely that - apart from its adaptive response to restore proteostasis - it also directly affects metabolic pathways. Here we investigate the direct role of the UPR in glucose homeostasis. O-GlcNAc is a post-translational modification that is highly responsive to glucose fluctuations. We find that UPR activation results in decreased O-GlcNAc modification, in line with reduced glucose metabolism. Our data indicate that UPR activation has no direct impact on the upstream processes in glucose metabolism; glucose transporter expression, glucose uptake and hexokinase activity. In contrast, prolonged UPR activation decreases glycolysis and mitochondrial metabolism. Decreased mitochondrial respiration is not accompanied by apoptosis or a structural change in mitochondria indicating that the reduction in metabolic rate upon UPR activation is a physiological non-apoptotic response. Metabolic decrease is prevented if the IRE1 pathway of the UPR is inhibited. This indicates that activation of IRE1 signaling induces a reduction in glucose metabolism, as part of an adaptive response. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Design information verification (DIV) of closed geological repositories (SAGOR activity 3c)

    International Nuclear Information System (INIS)

    Myatt, J.

    1998-02-01

    Following IAEA Advisory and Consultants Group meetings in September 1988 and in May 1991 respectively an IAEA multi-national Support Programme Task was initiated to consider the 'Development of Safeguards for Final Disposal of Spent Fuel in Geological Repositories' (SAGOR). A 'Technical Coordination Committee' (TCC) was set up with invited representatives from those Member State Support Programmes wishing to be involved. The joint programme, through the TCC, was given the task of studying the safeguards requirements in: conditioning plant (where the spent fuel is prepared for transfer to the repository); operating repositories (i.e. those in which the fuel is being emplaced); closed repositories. At the first meeting of the TCC in Washington in July 1994 the UK undertook to provide a study of the Design Information Verification (DIV) required in all three areas. For this activity the requirements, techniques and procedures for the Design Information Verification (DIV) of closed repositories have been considered. In completing the study the findings reported for activities 1b/c and 2c (descriptions of a Model Repository and Potential Diversion Paths, respectively) have been used in formulating any conclusions reached. It is also debatable as to whether this activity is strictly speaking DIV or is one of surveillance. As undeclared access can only be made to the emplacement areas of the repository by altering the physical makeup of the surrounding area; i.e. physically changing the 'design' of the surrounding area, however, this is deemed to be DIV. Although the techniques used appear to be those of surveillance they are being applied in this case as the tools of DIV. As with any facility there are a number of stages in its lifetime. For the purposes of this report the operating life of a repository is defined as being the time from inception to when it is finally decommissioned and sealed with the ground surface returned to being a green field. The repository is

  18. Efficient augmented Lagrangian-type preconditioning for the Oseen problem using Grad-Div stabilization

    KAUST Repository

    Heister, Timo

    2012-01-29

    Efficient preconditioning for Oseen-type problems is an active research topic. We present a novel approach leveraging stabilization for inf-sup stable discretizations. The Grad-Div stabilization shares the algebraic properties with an augmented Lagrangian-type term. Both simplify the approximation of the Schur complement, especially in the convection-dominated case. We exploit this for the construction of the preconditioner. Solving the discretized Oseen problem with an iterative Krylov-type method shows that the outer iteration numbers are retained independent of mesh size, viscosity, and finite element order. Thus, the preconditioner is very competitive. © 2012 John Wiley & Sons, Ltd.

  19. Efficient augmented Lagrangian-type preconditioning for the Oseen problem using Grad-Div stabilization

    KAUST Repository

    Heister, Timo; Rapin, Gerd

    2012-01-01

    Efficient preconditioning for Oseen-type problems is an active research topic. We present a novel approach leveraging stabilization for inf-sup stable discretizations. The Grad-Div stabilization shares the algebraic properties with an augmented Lagrangian-type term. Both simplify the approximation of the Schur complement, especially in the convection-dominated case. We exploit this for the construction of the preconditioner. Solving the discretized Oseen problem with an iterative Krylov-type method shows that the outer iteration numbers are retained independent of mesh size, viscosity, and finite element order. Thus, the preconditioner is very competitive. © 2012 John Wiley & Sons, Ltd.

  20. Muudatused tulekahjuhäiresüsteemide ehitamisel / Veiko Jürisson

    Index Scriptorium Estoniae

    Jürisson, Veiko, 1956-2008

    2005-01-01

    Tulekahju-signalisatsioonisüsteemi ehitusele eelneva kirjaliku nõusoleku taotlemisest kohalikult omavalitsuselt, valminud süsteemi üleandmisest omanikule ning automaatse tulekahjuhäiresüsteemi kasutusloa andmisest

  1. Cleareye In-Ground and In-Concrete DIV Inspections: FY11 Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Braatz, Brett G.; Tedeschi, Jonathan R.; Denslow, Kayte M.; Morra, Marino; Knopik, Clint D.; Severtsen, Ronald H.; Jones, Anthony M.; Lechelt, Wayne M.; McMakin, Douglas L.; Good, Morris S.; Sorensen, Jerry B.; Hall, Thomas E.

    2012-01-23

    This report summarizes the results of a series of feasibility testing studies for in-ground and in-concrete imaging/detection technologies including radar imaging and acoustic time-of flight method. The objectives of this project are: (1) Design Information Verification (DIV) Tools for In-Concrete Inspections - To determine the feasibility of using holographic radar imaging (HRI), radar imaging, and acoustic time-of-flight (TOF) non-destructive evaluation technologies to detect, locate and identify pipes and voids embedded in standard-density and high-density concrete walls that typify those the IAEA will need to verify during field inspections; (2) DIV Tools for In-Ground Inspections - To determine the feasibility of using HRI and radar imaging non-destructive evaluation technologies to detect, locate, and identify objects buried at various depths made of various materials (metal, plastic, wood, and concrete) and representing geometries that typify those the IAEA will need to verify during field inspections; and (3) Based on the results of the studies, recommend the next steps needed to realize fieldable tools for in-concrete and in-ground inspections (including detection of deeply buried polyvinyl chloride [PVC] pipes) that employ the technologies shown to be feasible.

  2. Re-localization of Cellular Protein SRp20 during Poliovirus Infection: Bridging a Viral IRES to the Host Cell Translation Apparatus

    Science.gov (United States)

    Fitzgerald, Kerry D.; Semler, Bert L.

    2011-01-01

    Poliovirus IRES-mediated translation requires the functions of certain canonical as well as non-canonical factors for the recruitment of ribosomes to the viral RNA. The interaction of cellular proteins PCBP2 and SRp20 in extracts from poliovirus-infected cells has been previously described, and these two proteins were shown to function synergistically in viral translation. To further define the mechanism of ribosome recruitment for the initiation of poliovirus IRES-dependent translation, we focused on the role of the interaction between cellular proteins PCBP2 and SRp20. Work described here demonstrates that SRp20 dramatically re-localizes from the nucleus to the cytoplasm of poliovirus-infected neuroblastoma cells during the course of infection. Importantly, SRp20 partially co-localizes with PCBP2 in the cytoplasm of infected cells, corroborating our previous in vitro interaction data. In addition, the data presented implicate the presence of these two proteins in viral translation initiation complexes. We show that in extracts from poliovirus-infected cells, SRp20 is associated with PCBP2 bound to poliovirus RNA, indicating that this interaction occurs on the viral RNA. Finally, we generated a mutated version of SRp20 lacking the RNA recognition motif (SRp20ΔRRM) and found that this protein is localized similar to the full length SRp20, and also partially co-localizes with PCBP2 during poliovirus infection. Expression of this mutated version of SRp20 results in a ∼100 fold decrease in virus yield for poliovirus when compared to expression of wild type SRp20, possibly via a dominant negative effect. Taken together, these results are consistent with a model in which SRp20 interacts with PCBP2 bound to the viral RNA, and this interaction functions to recruit ribosomes to the viral RNA in a direct or indirect manner, with the participation of additional protein-protein or protein-RNA interactions. PMID:21779168

  3. An approach to modeling and optimization of integrated renewable energy system (ires)

    Science.gov (United States)

    Maheshwari, Zeel

    The purpose of this study was to cost optimize electrical part of IRES (Integrated Renewable Energy Systems) using HOMER and maximize the utilization of resources using MATLAB programming. IRES is an effective and a viable strategy that can be employed to harness renewable energy resources to energize remote rural areas of developing countries. The resource- need matching, which is the basis for IRES makes it possible to provide energy in an efficient and cost effective manner. Modeling and optimization of IRES for a selected study area makes IRES more advantageous when compared to hybrid concepts. A remote rural area with a population of 700 in 120 households and 450 cattle is considered as an example for cost analysis and optimization. Mathematical models for key components of IRES such as biogas generator, hydropower generator, wind turbine, PV system and battery banks are developed. A discussion of the size of water reservoir required is also presented. Modeling of IRES on the basis of need to resource and resource to need matching is pursued to help in optimum use of resources for the needs. Fixed resources such as biogas and water are used in prioritized order whereas movable resources such as wind and solar can be used simultaneously for different priorities. IRES is cost optimized for electricity demand using HOMER software that is developed by the NREL (National Renewable Energy Laboratory). HOMER optimizes configuration for electrical demand only and does not consider other demands such as biogas for cooking and water for domestic and irrigation purposes. Hence an optimization program based on the need-resource modeling of IRES is performed in MATLAB. Optimization of the utilization of resources for several needs is performed. Results obtained from MATLAB clearly show that the available resources can fulfill the demand of the rural areas. Introduction of IRES in rural communities has many socio-economic implications. It brings about improvement in living

  4. SiDIVS: Simple Detection of Inductive Vehicle Signatures with a Multiplex Resonant Sensor

    Directory of Open Access Journals (Sweden)

    José J. Lamas-Seco

    2016-08-01

    Full Text Available This work provides a system capable of obtaining simultaneous inductive signatures of vehicles traveling on a roadway with minimal cost. Based on Time-Division Multiplexing (TDM with multiple oscillators, one for each inductive loop, the proposed system detects the presence of vehicles by means of a shift in the oscillation period of the selected loop and registers the signature of the detected vehicles by measuring the duration of a fixed number of oscillator pulses. In order to test the system in an actual environment, we implement a prototype that we denote as SiDIVS (Simple Detection of Inductive Vehicle Signatures and acquire different vehicle inductive signatures under real scenarios. We also test the robustness of the detector by simulating the effect of noise on the signature acquisition.

  5. The multi-targeted kinase inhibitor sorafenib inhibits enterovirus 71 replication by regulating IRES-dependent translation of viral proteins.

    Science.gov (United States)

    Gao, Meng; Duan, Hao; Liu, Jing; Zhang, Hao; Wang, Xin; Zhu, Meng; Guo, Jitao; Zhao, Zhenlong; Meng, Lirong; Peng, Yihong

    2014-06-01

    The activation of ERK and p38 signal cascade in host cells has been demonstrated to be essential for picornavirus enterovirus 71 (EV71) replication and up-regulation of virus-induced cyclooxygenase-2 (COX-2)/prostaglandins E2 (PGE2) expression. The aim of this study was to examine the effects of sorafenib, a clinically approved anti-cancer multi-targeted kinase inhibitor, on the propagation and pathogenesis of EV71, with a view to its possible mechanism and potential use in the design of therapy regimes for Hand foot and mouth disease (HFMD) patients with life threatening neurological complications. In this study, non-toxic concentrations of sorafenib were shown to inhibit the yield of infectious progeny EV71 (clinical BC08 strain) by about 90% in three different cell types. A similar inhibitory effect of sorafenib was observed on the synthesis of both viral genomic RNA and the VP1 protein. Interestingly, sorafenib exerted obvious inhibition of the EV71 internal ribosomal entry site (IRES)-mediated translation, the first step in picornavirus replication, by linking it to a firefly luciferase reporter gene. Sorafenib was also able to prevent both EV71-induced CPE and the activation of ERK and p38, which contributes to up-regulation COX-2/PGE2 expression induced by the virus. Overall, this study shows that sorafenib strongly inhibits EV71 replication at least in part by regulating viral IRES-dependent translation of viral proteins, indicating a novel potential strategy for the treatment of HFMD patients with severe neurological complications. To our knowledge, this is the first report that investigates the mechanism by which sorafenib inhibits EV71 replication. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Intestinal IRE1 Is Required for Increased Triglyceride Metabolism and Longer Lifespan under Dietary Restriction.

    Science.gov (United States)

    Luis, Nuno Miguel; Wang, Lifen; Ortega, Mauricio; Deng, Hansong; Katewa, Subhash D; Li, Patrick Wai-Lun; Karpac, Jason; Jasper, Heinrich; Kapahi, Pankaj

    2016-10-25

    Dietary restriction (DR) is one of the most robust lifespan-extending interventions in animals. The beneficial effects of DR involve a metabolic adaptation toward increased triglyceride usage. The regulatory mechanism and the tissue specificity of this metabolic switch remain unclear. Here, we show that the IRE1/XBP1 endoplasmic reticulum (ER) stress signaling module mediates metabolic adaptation upon DR in flies by promoting triglyceride synthesis and accumulation in enterocytes (ECs) of the Drosophila midgut. Consistently, IRE1/XBP1 function in ECs is required for increased longevity upon DR. We further identify sugarbabe, a Gli-like zinc-finger transcription factor, as a key mediator of the IRE1/XBP1-regulated induction of de novo lipogenesis in ECs. Overexpression of sugarbabe rescues metabolic and lifespan phenotypes of IRE1 loss-of-function conditions. Our study highlights the critical role of metabolic adaptation of the intestinal epithelium for DR-induced lifespan extension and explores the IRE1/XBP1 signaling pathway regulating this adaptation and influencing lifespan. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Animais, divórcio e consequências jurídicas

    Directory of Open Access Journals (Sweden)

    Camilo Henrique Silva

    2015-07-01

    Full Text Available http://dx.doi.org/10.5007/1807-1384.2015v12n1p102 O presente trabalho, por meio da pesquisa bibliográfica às leis, às decisões judiciais e à doutrina, tem por escopo trazer ao leitor, de maneira objetiva, as reflexões e o posicionamento do ordenamento jurídico brasileiro sobre o tema dos animais de estimação envolvidos em disputas de guarda no processo de divórcio. Nesse contexto, o escrito aborda a lei brasileira sobre guarda, direito de visita e alimentos, e sua utilização, por analogia, aos casos envolvendo animais de estimação, em razão da falta de norma específica. O texto retrata a tarefa dos magistrados e tribunais de se posicionarem nos casos levados a juízo e apresenta requisitos a serem observados para a decisão da lide, com vistas ao bem-estar e interesse do animal. Na disputa judicial pelos animais de estimação é legal a concessão da guarda e direito de visita aos cônjuges-tutores e a imposição de prestar alimentos ao animal.

  8. Analysis of Factors Affecting System Performance in the ASpIRE Challenge

    Science.gov (United States)

    2015-12-13

    performance in the ASpIRE ( Automatic Speech recognition In Reverberant Environments) challenge. In particular, overall word error rate (WER) of the solver...in mismatched conditions. Index Terms: speech recognition, reverberant rooms, microphone audio 1. Introduction The development of automatic ...IEEE Workshop on Automatic Speech Recognition and Understanding, 2005. [7] Harper, M., The Automatic Speech Recognition in Reverberant

  9. Inflammatory conditions induce IRES-dependent translation of cyp24a1.

    Directory of Open Access Journals (Sweden)

    Daniela Rübsamen

    Full Text Available Rapid alterations in protein expression are commonly regulated by adjusting translation. In addition to cap-dependent translation, which is e.g. induced by pro-proliferative signaling via the mammalian target of rapamycin (mTOR-kinase, alternative modes of translation, such as internal ribosome entry site (IRES-dependent translation, are often enhanced under stress conditions, even if cap-dependent translation is attenuated. Common stress stimuli comprise nutrient deprivation, hypoxia, but also inflammatory signals supplied by infiltrating immune cells. Yet, the impact of inflammatory microenvironments on translation in tumor cells still remains largely elusive. In the present study, we aimed at identifying translationally deregulated targets in tumor cells under inflammatory conditions. Using polysome profiling and microarray analysis, we identified cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase to be translationally upregulated in breast tumor cells co-cultured with conditioned medium of activated monocyte-derived macrophages (CM. Using bicistronic reporter assays, we identified and validated an IRES within the 5' untranslated region (5'UTR of cyp24a1, which enhances translation of cyp24a1 upon CM treatment. Furthermore, IRES-dependent translation of cyp24a1 by CM was sensitive to phosphatidyl-inositol-3-kinase (PI3K inhibition, while constitutive activation of Akt sufficed to induce its IRES activity. Our data provide evidence that cyp24a1 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment. Considering the negative feedback impact of cyp24a1 on the vitamin D responses, the identification of a novel, translational mechanism of cyp24a1 regulation might open new possibilities to overcome the current limitations of vitamin D as tumor therapeutic option.

  10. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon

    Directory of Open Access Journals (Sweden)

    Moes Lorin

    2007-11-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV and the more distantly related Hepatitis C virus (HCV pseudoknot function in translation has been demonstrated. Results To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical

  11. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon.

    Science.gov (United States)

    Moes, Lorin; Wirth, Manfred

    2007-11-22

    Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated. To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the

  12. Switch from cap- to factorless IRES-dependent 0 and +1 frame translation during cellular stress and dicistrovirus infection.

    Directory of Open Access Journals (Sweden)

    Qing S Wang

    Full Text Available Internal ribosome entry sites (IRES are utilized by a subset of cellular and viral mRNAs to initiate translation during cellular stress and virus infection when canonical cap-dependent translation is compromised. The intergenic region (IGR IRES of the Dicistroviridae uses a streamlined mechanism in which it can directly recruit the ribosome in the absence of initiation factors and initiates translation using a non-AUG codon. A subset of IGR IRESs including that from the honey bee viruses can also direct translation of an overlapping +1 frame gene. In this study, we systematically examined cellular conditions that lead to IGR IRES-mediated 0 and +1 frame translation in Drosophila S2 cells. Towards this, a novel bicistronic reporter that exploits the 2A "stop-go" peptide was developed to allow the detection of IRES-mediated translation in vivo. Both 0 and +1 frame translation by the IGR IRES are stimulated under a number of cellular stresses and in S2 cells infected by cricket paralysis virus, demonstrating a switch from cap-dependent to IRES-dependent translation. The regulation of the IGR IRES mechanism ensures that both 0 frame viral structural proteins and +1 frame ORFx protein are optimally expressed during virus infection.

  13. Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α3DIV, that Sequesters Toxic Heavy Metals.

    Science.gov (United States)

    Plegaria, Jefferson S; Dzul, Stephen P; Zuiderweg, Erik R P; Stemmler, Timothy L; Pecoraro, Vincent L

    2015-05-12

    De novo protein design is a biologically relevant approach that provides a novel process in elucidating protein folding and modeling the metal centers of metalloproteins in a completely unrelated or simplified fold. An integral step in de novo protein design is the establishment of a well-folded scaffold with one conformation, which is a fundamental characteristic of many native proteins. Here, we report the NMR solution structure of apo α3DIV at pH 7.0, a de novo designed three-helix bundle peptide containing a triscysteine motif (Cys18, Cys28, and Cys67) that binds toxic heavy metals. The structure comprises 1067 NOE restraints derived from multinuclear multidimensional NOESY, as well as 138 dihedral angles (ψ, φ, and χ1). The backbone and heavy atoms of the 20 lowest energy structures have a root mean square deviation from the mean structure of 0.79 (0.16) Å and 1.31 (0.15) Å, respectively. When compared to the parent structure α3D, the substitution of Leu residues to Cys enhanced the α-helical content of α3DIV while maintaining the same overall topology and fold. In addition, solution studies on the metalated species illustrated metal-induced stability. An increase in the melting temperatures was observed for Hg(II), Pb(II), or Cd(II) bound α3DIV by 18-24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α3DIV produced an average Hg(II)-S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α3DIV reveals an asymmetric distorted triscysteine metal binding site, which offers a model for native metalloregulatory proteins with thiol-rich ligands that function in regulating toxic heavy metals, such as ArsR, CadC, MerR, and PbrR.

  14. Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis

    Directory of Open Access Journals (Sweden)

    Aseem Pandey

    2018-04-01

    Full Text Available Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR sensor IRE1α (inositol-requiring enzyme 1 and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1 conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.

  15. As bases eleitorais de Lula e do PT: do distanciamento ao divórcio

    Directory of Open Access Journals (Sweden)

    Sonia Luiza Terron

    2010-11-01

    Full Text Available O debate sobre o distanciamento entre Lula e o Partido dos Trabalhadores (PT ganhou corpo na eleição de 2006, quando a popularidade de Lula e de seu governo não se estendeu ao PT, como seria esperado. Pela primeira vez o partido não melhorou o desempenho na votação para a Câmara dos Deputados em relação à eleição anterior. Analisamos as bases eleitorais de Lula e do PT nas quatro últimas eleições para presidente e deputado federal (1994-2006, e comprovamos o distanciamento entre elas. Pesquisamos a distribuição espacial das votações ao nível do município com mapas, estatísticas e regressões espaciais. Identificamos o "descolamento" dos territórios eleitorais do candidato e do partido a cada eleição, e as regressões espaciais mediram a crescente independência espaço-temporal das votações: os coeficientes baixaram de 0,85 em 1994, para 0,15 em 2006. Houve um processo de distanciamento geossocial, no período de 1994 a 2002, e um "divórcio" entre as bases eleitorais, em 2006, quando se formaram territórios de configurações praticamente irrelacionadas.The debate about the distancing between Lula and the Workers Party (PT gained acceptance in the 2006 election, when the popularity of Lula and his government did not extend to the PT, as expected. For the first time the party did not improve performance in the voting for the Chamber of Deputies regarding the previous election. We analyzed the electoral bases of Lula and the PT in the last four elections for president and federal deputies (1994-2006, and we prove the distancing between them. We studied the spatial distribution of votes at the municipal level with maps, statistics and spatial regression. We identified the detachment of the candidate's and party's electoral territories every election, and the regressions measured the growing spatial-temporal independence of the votings: the coefficient fell from 0.85 in 1994 to 0.15 in 2006. There was a process of

  16. Generation of Pax6-IRES-EGFP knock-in mouse via the cloning-free CRISPR/Cas9 system to reliably visualize neurodevelopmental dynamics.

    Science.gov (United States)

    Inoue, Yukiko U; Morimoto, Yuki; Hoshino, Mikio; Inoue, Takayoshi

    2018-07-01

    Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, neocortical arealization, and so forth. Visualization of Pax6 expression dynamics in these events could offer numerous advantages to neurodevelopmental studies. While CRISPR/Cas9 system has dramatically accelerated one-step generation of knock-out mouse, establishment of gene-cassette knock-in mouse via zygote injection has been considered insufficient due to its low efficiency. Recently, an improved CRISPR/Cas9 system for effective gene-cassette knock-in has been reported, where the native form of guide RNAs (crRNA and tracrRNA) assembled with recombinant Cas9 protein are directly delivered into mouse fertilized eggs. Here we apply this strategy to insert IRES-EGFP-pA cassette into Pax6 locus and achieve efficient targeted insertions of the 1.8 kb reporter gene. In Pax6-IRES-EGFP mouse we have generated, EGFP-positive cells reside in the eyes and cerebellum as endogenous Pax6 expressing cells at postnatal day 2. At the early embryonic stages when the embryos are transparent, EGFP-positive regions can be easily identified without PCR-based genotyping, precisely recapitulating the endogenous Pax6 expression patterns. Remarkably, at E12.5, the graded expression patterns of Pax6 in the developing neocortex now become recognizable in our knock-in mice, serving a sufficiently sensitive and useful tool to precisely visualize neurodevelopmental processes. Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.

  17. Role of IRE1α/XBP-1 in Cystic Fibrosis Airway Inflammation

    Directory of Open Access Journals (Sweden)

    Carla M. P. Ribeiro

    2017-01-01

    Full Text Available Cystic fibrosis (CF pulmonary disease is characterized by chronic airway infection and inflammation. The infectious and inflamed CF airway environment impacts on the innate defense of airway epithelia and airway macrophages. The CF airway milieu induces an adaptation in these cells characterized by increased basal inflammation and a robust inflammatory response to inflammatory mediators. Recent studies have indicated that these responses depend on activation of the unfolded protein response (UPR. This review discusses the contribution of airway epithelia and airway macrophages to CF airway inflammatory responses and specifically highlights the functional importance of the UPR pathway mediated by IRE1/XBP-1 in these processes. These findings suggest that targeting the IRE1/XBP-1 UPR pathway may be a therapeutic strategy for CF airway disease.

  18. Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE

    Directory of Open Access Journals (Sweden)

    Kos Bor

    2015-09-01

    Full Text Available Background. Irreversible electroporation (IRE is a tissue ablation method, which relies on the phenomenon of electroporation. When cells are exposed to a sufficiently electric field, the plasma membrane is disrupted and cells undergo an apoptotic or necrotic cell death. Although heating effects are known IRE is considered as non-thermal ablation technique and is currently applied to treat tumors in locations where thermal ablation techniques are contraindicated.

  19. Changes of hyoid bone position following treatment of class II div1 malocclusion with Farmand functional appliance

    Directory of Open Access Journals (Sweden)

    Yassaei S

    2007-01-01

    Full Text Available Background and Aim: Unlike other bones of the head and neck, hyoid bone has no bony articulations. It is connected to mandible, cranium and pharynx through muscles and ligaments. During treatment with functional appliance in patients with class II div1 malocclusion, mandible is positioned in inferior and anterior direction. Regarding the relation between hyoid and mandibular bone, alterations of hyoid bone position can be a result of functional appliance therapy. The aim of this study was to evaluate the changes of hyoid bone position following treatment with Farmand functional appliance in patients with class II div 1 malocclusion. Materials and Methods: In this before-after clinical trial, 28 patients with class II div 1 malocclusion which were under treatment with Farmand functional appliance for 11 months were selected. Facial growth in vertical, normal or horizontal direction was determined by cephalometric measurement. Data were analyzed with Paired-t test to compare the differences of mean values pre and post treatment. Variance analysis was used to compare the three growth patterns. P<0.05 was considered as the limit of significance. Results: Hyoid bone shifted significantly forward in horizontal dimension (P<0.01 and non-significantly upward in vertical dimension. There was no significant difference among the three studied groups with respect to hyoid bone position alterations in horizontal dimension but significant difference was observed between horizontal and vertical growth pattern in vertical dimension (P<0.05. There was significant correlation between decrease of ANB angle and forward movement of hyoid bone. Conclusion: Based on the results of this study, treatment with Farmand functional appliance (Fa II leads to significant alterations in the position and anterior displacement of the hyoid bone.

  20. The use of IRE in multi-modality treatment for oligometastatic pancreatic cancer.

    Science.gov (United States)

    Hong, Young; Rice, Jonathan; Sharma, Divyansh; Martin, Robert C G

    2018-03-02

    Pancreatic ductal adenocarcinoma (PDAC) often presents late with only 20% of patients being candidates for resection while majority already have advanced metastases with median overall survival of 3-6 months. Currently, the role of oligometastasectomy and local therapy options in PDAC is unknown in patients who have favorable response to systemic chemotherapy. The aim of this study is to analyze the survival outcome of oligometastasectomy and local IRE therapy in select patients who are treated with systemic chemotherapy for PDAC metastases. We utilized a prospective database from 2010 to 2016 to identify patients with local surgical therapy after induction systemic chemotherapy for oligometastatic PDAC (Stage 4). The initial local therapy treatment of distant metastatic lesions was followed by adjuvant chemotherapy. Subsequently, resection of the primary PDAC in conjunction with irreversible electroporation (IRE) was performed after favorable response by RECIST criteria. Seven patients were identified with metastatic PDAC treated with oligometastasectomy and/or local therapy. There was single metastatic lesion in 43% (3/7) of which 57% (4/7) were localized in the liver. The treatment of the primary pancreatic cancer was performed utilizing IRE in situ in 6/7 (86%) of patients in our study with resection or radiation of oligometastasis. The median survival in our study group was 16 months with 28% (2/7) patients who remain NED (range 16-41 months). Combination of systemic chemotherapy and oligometastasectomy with adjunctive local IRE therapy is a feasible treatment strategy in highly select patients with oligometastatic PDAC that demonstrate favorable tumor biology with objective response to systemic therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Single step production of Cas9 mRNA for zygote injection.

    Science.gov (United States)

    Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

    2018-03-01

    Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

  2. Divórcio e funcionamento psicológico em adultos e crianças

    OpenAIRE

    Silva, Diogo Jorge Pereira do Vale Lamela da

    2013-01-01

    Tese de doutoramento em Psicologia (área de especialização de Psicologia Clínica) A presente dissertação foi orientada por dois enquadramentos teóricos: o modelo de Desenvolvimento Positivo da Personalidade na idade adulta (Staudinger & Bowen, 2010) e no modelo ecológico da coparentalidade (Feinberg, 2003). Objetivo: Este trabalho pretendeu responder a quatro objetivos. (1) Identificar variáveis sócio-demográficas, características do divórcio e indicadores personológicos qu...

  3. An internal ribosome entry site directs translation of the 3'-gene from Pelargonium flower break virus genomic RNA: implications for infectivity.

    Directory of Open Access Journals (Sweden)

    Olga Fernández-Miragall

    Full Text Available Pelargonium flower break virus (PFBV, genus Carmovirus has a single-stranded positive-sense genomic RNA (gRNA which contains five ORFs. The two 5'-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3'-proximal ORF encodes a polypeptide (p37 which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5'-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3'-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES. Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.

  4. An internal ribosome entry site directs translation of the 3'-gene from Pelargonium flower break virus genomic RNA: implications for infectivity.

    Science.gov (United States)

    Fernández-Miragall, Olga; Hernández, Carmen

    2011-01-01

    Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 5'-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3'-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5'-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3'-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.

  5. Translational control of ceruloplasmin gene expression: Beyond the IRE

    Directory of Open Access Journals (Sweden)

    BARSANJIT MAZUMDER

    2006-01-01

    Full Text Available Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor; erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5'-untranslated region (UTR; and 15-lipoxygenase is regulated by specific proteins binding to the 3'-UTR. Ceruloplasmin (Cp is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN-γ in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT, to a specific GAIT element in the Cp 3'-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

  6. Accurate macromolecular crystallographic refinement: incorporation of the linear scaling, semiempirical quantum-mechanics program DivCon into the PHENIX refinement package

    Energy Technology Data Exchange (ETDEWEB)

    Borbulevych, Oleg Y.; Plumley, Joshua A.; Martin, Roger I. [QuantumBio Inc., 2790 West College Avenue, State College, PA 16801 (United States); Merz, Kenneth M. Jr [University of Florida, Gainesville, Florida (United States); Westerhoff, Lance M., E-mail: lance@quantumbioinc.com [QuantumBio Inc., 2790 West College Avenue, State College, PA 16801 (United States)

    2014-05-01

    Semiempirical quantum-chemical X-ray macromolecular refinement using the program DivCon integrated with PHENIX is described. Macromolecular crystallographic refinement relies on sometimes dubious stereochemical restraints and rudimentary energy functionals to ensure the correct geometry of the model of the macromolecule and any covalently bound ligand(s). The ligand stereochemical restraint file (CIF) requires a priori understanding of the ligand geometry within the active site, and creation of the CIF is often an error-prone process owing to the great variety of potential ligand chemistry and structure. Stereochemical restraints have been replaced with more robust functionals through the integration of the linear-scaling, semiempirical quantum-mechanics (SE-QM) program DivCon with the PHENIX X-ray refinement engine. The PHENIX/DivCon package has been thoroughly validated on a population of 50 protein–ligand Protein Data Bank (PDB) structures with a range of resolutions and chemistry. The PDB structures used for the validation were originally refined utilizing various refinement packages and were published within the past five years. PHENIX/DivCon does not utilize CIF(s), link restraints and other parameters for refinement and hence it does not make as many a priori assumptions about the model. Across the entire population, the method results in reasonable ligand geometries and low ligand strains, even when the original refinement exhibited difficulties, indicating that PHENIX/DivCon is applicable to both single-structure and high-throughput crystallography.

  7. Small interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes

    Directory of Open Access Journals (Sweden)

    Dash Srikanta

    2006-11-01

    Full Text Available Abstract Background The antiviral action of interferon alpha targets the 5' untranslated region (UTR used by hepatitis C virus (HCV to translate protein by an internal ribosome entry site (IRES mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Results Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. Conclusion These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

  8. Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

    Science.gov (United States)

    Andreucci, Elena; Bianchini, Francesca; Biagioni, Alessio; Del Rosso, Mario; Papucci, Laura; Schiavone, Nicola; Magnelli, Lucia

    2017-01-01

    Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features. FGF2 is an alternative translation product expressed as different isoforms termed LMW and HMW. FGF2 is involved in melanoma development and progression. HMW FGF2 isoforms enhance in vitro motility of melanoma cells. LMW FGF2 confers stem-like features and increases in vivo metastasization. LMW FGF2 promotes angiogenesis while HMW FGF2 induces vasculogenic mimicry.

  9. Investigation of the Stress Intensity Limits of ASME Section III Div.5 for Structure Design Criteria of SFR Fuel Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Choo, Jin-Yup; Kim, Hyung-Kyu; Cheon, Jin-Sik [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2016-10-15

    These affect the mechanical design of the fuel assembly components. And thus, appropriate structural design criteria should also be chosen to incorporate the specific design conditions of the SFR fuel assemblies. Among them, the temperature is one of the most crucial conditions to be concerned because the sodium coolant temperature is normally more than 500ºC which is much higher than that of the LWR (< 350ºC). This implies that a thermal creep should be significantly considered in the SFR fuel assembly mechanical design. In addition to the high temperature condition, an irradiation swelling is also an important behavior that the SFR fuel assembly material should accommodate. To incorporate the temperature and irradiation impacts, the material of the fuel assembly components is presently determined to be made of HT-9, the ferriticmartensitic steel. In this paper, the ASME Sec. III Div. 5 (referred to as ‘Div. 5’ hereinafter), which was developed for a ‘high temperature reactor’, is considered as one of the structural design criteria for the mechanical design of SFR fuel assemblies. In this paper, the stress intensity limits, S{sub m} and S{sub t} of HT-9 were built for the structural criteria of an SFR fuel assembly. S{sub m} is obtained from the ultimate strength. As for S{sub t}, it is more complicated because of its dependency of time duration in addition to temperature. Following the definition of S{sub mt}, the method in the ASME Sec. III Div. 1, Subsec. NH was consulted. We found that the Sm is adopted as S{sub mt} under the temperature about 470ºC which is relatively low temperature range and over 470ºC with relatively short time duration as 1000 hours. And the S{sub t} is adopted as Smt at over 470ºC and long time duration over 34800 hours, and over 520ºC and 10{sup 4} hours too. And at over 570ºC and 1000 hours, and at over 630ºC and 100 hours, S{sub t} is also adopted for S{sub mt}.

  10. The metabolic ER stress sensor IRE1α suppresses alternative activation of macrophages and impairs energy expenditure in obesity.

    Science.gov (United States)

    Shan, Bo; Wang, Xiaoxia; Wu, Ying; Xu, Chi; Xia, Zhixiong; Dai, Jianli; Shao, Mengle; Zhao, Feng; He, Shengqi; Yang, Liu; Zhang, Mingliang; Nan, Fajun; Li, Jia; Liu, Jianmiao; Liu, Jianfeng; Jia, Weiping; Qiu, Yifu; Song, Baoliang; Han, Jing-Dong J; Rui, Liangyou; Duan, Sheng-Zhong; Liu, Yong

    2017-05-01

    Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1 f/f ; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1 f/f ; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.

  11. Interdisciplinary management of an adult patient with class II div 1 Malocclusion, Anterior Open Bite and multiple missing molars

    Directory of Open Access Journals (Sweden)

    Rohit Mehrotra

    2014-01-01

    Full Text Available This case report describes the esthetic and functional rehabilitation of a female patient in her mid forties having Class II Div 1 malocclusion, anterior open bite and multiple missing molars, with orthodontics and implant prosthodontics. The patient had bilaterally missing upper first and third molars and lower first and second molars. Orthodontic treatment using skeletal anchorage was performed to retract and align the upper anteriors and correct the open bite. In the upper arch, first molar spaces were closed and no prosthesis was needed. In the lower arch, the anteriors and premolars were aligned and implants were placed bilaterally to replace the missing molars and provide proper occlusion with the upper posteriors. Significant improvement in the occlusion, smile and facial esthetics was achieved. This article highlights the importance of an interdisciplinary team approach for providing optimum treatment to many adult patients.

  12. Accurate macromolecular crystallographic refinement: incorporation of the linear scaling, semiempirical quantum-mechanics program DivCon into the PHENIX refinement package.

    Science.gov (United States)

    Borbulevych, Oleg Y; Plumley, Joshua A; Martin, Roger I; Merz, Kenneth M; Westerhoff, Lance M

    2014-05-01

    Macromolecular crystallographic refinement relies on sometimes dubious stereochemical restraints and rudimentary energy functionals to ensure the correct geometry of the model of the macromolecule and any covalently bound ligand(s). The ligand stereochemical restraint file (CIF) requires a priori understanding of the ligand geometry within the active site, and creation of the CIF is often an error-prone process owing to the great variety of potential ligand chemistry and structure. Stereochemical restraints have been replaced with more robust functionals through the integration of the linear-scaling, semiempirical quantum-mechanics (SE-QM) program DivCon with the PHENIX X-ray refinement engine. The PHENIX/DivCon package has been thoroughly validated on a population of 50 protein-ligand Protein Data Bank (PDB) structures with a range of resolutions and chemistry. The PDB structures used for the validation were originally refined utilizing various refinement packages and were published within the past five years. PHENIX/DivCon does not utilize CIF(s), link restraints and other parameters for refinement and hence it does not make as many a priori assumptions about the model. Across the entire population, the method results in reasonable ligand geometries and low ligand strains, even when the original refinement exhibited difficulties, indicating that PHENIX/DivCon is applicable to both single-structure and high-throughput crystallography.

  13. Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Saioa Márquez

    2017-06-01

    Full Text Available Human monocyte-derived dendritic cells (DCs exposed to pathogen-associated molecular patterns (PAMPs undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.

  14. Gamma background radiation monitoring results (1990-95) in the environs around IRE OSCOM, Orissa using TLDs

    Energy Technology Data Exchange (ETDEWEB)

    Mehta, N K; Bapat, V N; Nambi, K S.V. [Bhabha Atomic Research Centre, Mumbai (India). Environmental Assessment Div.; Saha, S C [Bhabha Atomic Research Centre, Bombay (India). Health Physics Div.

    1997-09-01

    Quarterly environmental radiation monitoring data using TLDs in about 28 locations in and around IRE-OSCOM covering a six year period 1990-95 is presented in the report. While the indoor radiation levels in beach areas having monazite deposits recorded annual average of 653{+-}230 mR/y, that in IRE residential colony gave a value of 240 {+-}30 mR/y. The radiation levels recorded inside the OSCOM plant are well within the limits prescribed for occupational radiation exposures. (author). 3 refs., 7 figs., 1 tab.

  15. Gamma background radiation monitoring results (1990-95) in the environs around IRE OSCOM, Orissa using TLDs

    International Nuclear Information System (INIS)

    Mehta, N.K.; Bapat, V.N.; Nambi, K.S.V.; Saha, S.C.

    1997-01-01

    Quarterly environmental radiation monitoring data using TLDs in about 28 locations in and around IRE-OSCOM covering a six year period 1990-95 is presented in the report. While the indoor radiation levels in beach areas having monazite deposits recorded annual average of 653±230 mR/y, that in IRE residential colony gave a value of 240 ±30 mR/y. The radiation levels recorded inside the OSCOM plant are well within the limits prescribed for occupational radiation exposures. (author). 3 refs., 7 figs., 1 tab

  16. Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

    Science.gov (United States)

    Back, Sung Hoon; Kim, Yoon Ki; Kim, Woo Jae; Cho, Sungchan; Oh, Hoe Rang; Kim, Jung-Eun; Jang, Sung Key

    2002-01-01

    The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA. PMID:11836431

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

    Science.gov (United States)

    Hidema, Shizu; Fukuda, Tomokazu; Hiraoka, Yuichi; Mizukami, Hiroaki; Hayashi, Ryotaro; Otsuka, Ayano; Suzuki, Shingo; Miyazaki, Shinji; Nishimori, Katsuhiko

    2016-05-01

    The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. © 2015 Wiley Periodicals, Inc.

  20. Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE).

    Science.gov (United States)

    Kos, Bor; Voigt, Peter; Miklavcic, Damijan; Moche, Michael

    2015-09-01

    Irreversible electroporation (IRE) is a tissue ablation method, which relies on the phenomenon of electroporation. When cells are exposed to a sufficiently electric field, the plasma membrane is disrupted and cells undergo an apoptotic or necrotic cell death. Although heating effects are known IRE is considered as non-thermal ablation technique and is currently applied to treat tumors in locations where thermal ablation techniques are contraindicated. The manufacturer of the only commercially available pulse generator for IRE recommends a voltage-to-distance ratio of 1500 to 1700 V/cm for treating tumors in the liver. However, major blood vessels can influence the electric field distribution. We present a method for treatment planning of IRE which takes the influence of blood vessels on the electric field into account; this is illustrated on a treatment of 48-year-old patient with a metastasis near the remaining hepatic vein after a right side hemi-hepatectomy. Output of the numerical treatment planning method shows that a 19.9 cm3 irreversible electroporation lesion was generated and the whole tumor was covered with at least 900 V/cm. This compares well with the volume of the hypodense lesion seen in contrast enhanced CT images taken after the IRE treatment. A significant temperature raise occurs near the electrodes. However, the hepatic vein remains open after the treatment without evidence of tumor recurrence after 6 months. Treatment planning using accurate computer models was recognized as important for electrochemotherapy and irreversible electroporation. An important finding of this study was, that the surface of the electrodes heat up significantly. Therefore the clinical user should generally avoid placing the electrodes less than 4 mm away from risk structures when following recommendations of the manufacturer.

  1. Irreversible Electroporation (IRE) Fails to Demonstrate Efficacy in a Prospective Multicenter Phase II Trial on Lung Malignancies: The ALICE Trial

    Energy Technology Data Exchange (ETDEWEB)

    Ricke, Jens, E-mail: jens.ricke@med.ovgu.de; Jürgens, Julian H. W., E-mail: julian.juergens@med.ovgu.de [University of Magdeburg, Department of Radiology and Nuclear Medicine (Germany); Deschamps, Frederic; Tselikas, Lambros [Institut de Cancérologie Gustave Roussy, Department of Image Guided Therapy (France); Uhde, Katja; Kosiek, Ortrud [University of Magdeburg, Department of Radiology and Nuclear Medicine (Germany); Baere, Thierry De [Institut de Cancérologie Gustave Roussy, Department of Image Guided Therapy (France)

    2015-04-15

    PurposeTo assess safety and efficacy of irreversible electroporation (IRE) of lung malignancies.Materials and MethodsPatients with primary and secondary lung malignancies and preserved lung function were included in this prospective single arm trial. Primary and secondary endpoints were safety and efficacy. Recruitment goal was 36 subjects in 2 centers. Patients underwent IRE under general anesthesia with probe placement performed in Fluoroscopy-CT. The IRE system employed was NanoKnife{sup ®} (Angiodynamics). System settings for the ablation procedure followed the manufacturer’s recommendations. The Mann–Whitney U test was used to evaluate the correlation of nine technical parameters with local tumor control. Median follow up was 12 months.ResultsThe expected efficacy was not met at interim analysis and the trial was stopped prematurely after inclusion of 23 patients (13/10 between both centers). The dominant tumor entity was colorectal (n = 13). The median tumor diameter was 16 mm (8–27 mm). Pneumothoraces were observed in 11 of 23 patients with chest tubes required in 8 (35 %). Frequently observed alveolar hemorrhage never led to significant hemoptysis. 14/23 showed progressive disease (61 %). Stable disease was found in 1 (4 %), partial remission in 1 (4 %) and complete remission in 7 (30 %) patients. The relative increase of the current during ablation was significantly higher in the group treated successfully as compared to the group presenting local recurrence (p < 0.05). Needle tract seeding was found in three cases (13 %).ConclusionsIRE is not effective for the treatment of lung malignancies. We hypothesize that the energy deposition with current IRE probes is highly sensitive to air exposure.

  2. Investigation of the Stress Intensity Limits of ASME Section III Div.5 for Structure Design Criteria of SFR Fuel Assembly

    International Nuclear Information System (INIS)

    Choo, Jin-Yup; Kim, Hyung-Kyu; Cheon, Jin-Sik

    2017-01-01

    In this paper, the stress intensity limits, Sm and St of HT-9 were built for the structural criteria of an SFR fuel assembly. Sm is obtained from the ultimate strength. As for St, it is more complicated because of its dependency of time duration in addition to temperature. Following the definition of Smt, the method in the ASME Sec. III Div. 1, Subsec. NH was consulted. We found that the Sm is adopted as Smt under the temperature about 470 .deg. C which is relatively low temperature range and over 470 .deg. C with relatively short time duration as 1000 hours. And the St is adopted as Smt at over 470 .deg. C and long time duration over 34800 hours, and over 520 .deg. C and 104 hours too. And at over 570 .deg. C and 1000 hours, and at over 630 .deg. C and 100 hours, St is also adopted for Smt. To use the present result as design criteria, a stringent examination needs to be carried out, because those are calculated from the formulae of HT-9 without an experimental validation. Therefore, an experimental work on the mechanical properties of HT-9 will be necessary.

  3. As Bruxas no Divã: o desafio de ser criativo e ousar no ensino de artes

    Directory of Open Access Journals (Sweden)

    Marcelo Rossa

    2012-11-01

    Este artigo relata o projeto “As Bruxas no Divã”, que consiste na produção de um filme no Instituto Estadual de Educação (IEE escola da rede estadual de ensino de SC. Nele introduziu-se a linguagem cinematográfica na Educação Infantil, contribuindo no processo de ensino e aprendizagem de Artes. O objetivo deste projeto foi desenvolver uma vivência dentro das diversas linguagens artísticas, bem como aguçar a reflexão sobre questões existenciais da vida. O projeto oportunizou a utilização de diversas linguagens artísticas bem como as novas tecnologias. A motivação para seu desenvolvimento foi à literatura e a produção de cinema amador infantil e se mostraram uma forma de comunicação apta a estabelecer interdisciplinaridade de várias áreas do conhecimento de forma criativa e harmoniosa.

  4. HIV-1 transcripts use IRES-initiation under conditions where Cap-dependent translation is restricted by poliovirus 2A protease.

    Directory of Open Access Journals (Sweden)

    Raquel Amorim

    Full Text Available The 30 different species of mRNAs synthesized during the HIV-1 replication cycle are all capped and polyadenilated. Internal ribosome entry sites have been recognized in the 5' untranslated region of some mRNA species of HIV-1, which would contribute to an alternative mechanism of initiation of mRNA translation. However, the Cap-dependent translation is assumed to be the main mechanism driving the initiation of HIV-1 protein synthesis. In this work, we describe a cell system in which lower to higher levels of transient expression of the poliovirus 2A protease strongly inhibited cellular Cap-dependent translation with no toxic effect to the cells during a 72-hour time frame. In this system, the synthesis of HIV-1 proteins was inhibited in a temporal dose-dependent way. Higher levels of 2A protease expression severely inhibited HIV-1 protein synthesis during the first 24 hours of infection consequently inhibiting viral production and infectivity. Intermediate to lower levels of 2A Protease expression caused the inhibition of viral protein synthesis only during the first 48 hours of viral replication. After this period both protein synthesis and viral release were recovered to the control levels. However, the infectivity of viral progeny was still partially inhibited. These results indicate that two mechanisms of mRNA translation initiation contribute to the synthesis of HIV-1 proteins; during the first 24-48 hours of viral replication HIV-1 protein synthesis is strongly dependent on Cap-initiation, while at later time points IRES-driven translation initiation is sufficient to produce high amounts of viral particles.

  5. Irreversible electroporation ablation (IRE of unresectable soft tissue tumors: learning curve evaluation in the first 150 patients treated.

    Directory of Open Access Journals (Sweden)

    Prejesh Philips

    Full Text Available BACKGROUND: Irreversible electroporation (IRE is a novel technology that uses peri-target discrete probes to deliver high-voltage localized electric current to induce cell death without thermal-induced coagulative necrosis. "Learnability" and consistently effective results by novice practitioners is essential for determining acceptance of novel techniques. This multi-center prospectively-collected database study evaluates the learning curve of IRE. METHODS: Analysis of 150 consecutive patients over 7 institutions from 9/2010-7/2012 was performed with patients treated divided into 3 groups A (1(st 50 patients treated, B (2(nd 50 and C (3(rd 50 patients treated chronologically and analyzed for outcomes. RESULTS: A total of 167 IRE procedures were performed, with a majority being liver(39.5% and pancreatic(35.5% lesions. The three groups were similar with respect to co-morbidities and demographics. Group C had larger lesions (3.9 vs 3 cm,p=0.001, more numerous lesions (3.2 vs 2.2,p=0.07, more vascular invasion(p=0.001, underwent more associated procedures(p=0.001 and had longer operative times(p<0.001. Despite this, they had similar complication and high-grade complication rates(p=0.24. Attributable morbidity rate was 13.3%(total 29.3% and high-grade complications were seen in 4.19%(total 12.6%. Pancreatic lesions(p=0.001 and laparotomy(p=0.001 were associated with complications. CONCLUSION: The review represents that single largest review of IRE soft tissue ablation demonstrating initial patient selection and safety. Over time, complex treatments of larger lesions and lesions with greater vascular involvement were performed without a significant increase in adverse effects or impact on local relapse free survival. This evolution demonstrates the safety profile of IRE and speed of graduation to more complex lesions, which was greater than 5 cases by institution. IRE is a safe and effective alternative to conventional ablation with a demonstrable

  6. Septal membrane localization by C-terminal amphipathic α-helices of MinD in Bacillus subtilis mutant cells lacking MinJ or DivIVA.

    Science.gov (United States)

    Ishikawa, Kazuki; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

    2017-10-18

    The Min system, which inhibits assembly of the cytokinetic protein FtsZ, is largely responsible for positioning the division site in rod-shaped bacteria. It has been reported that MinJ, which bridges DivIVA and MinD, is targeted to the cell poles by an interaction with DivIVA, and that MinJ in turn recruits MinCD to the cell poles. MinC, however, is located primarily at active division sites at mid-cell when expressed from its native promoter. Surprisingly, we found that Bacillus subtilis MinD is located at nascent septal membranes and at an asymmetric site on lateral membranes between nascent septal membranes in filamentous cells lacking MinJ or DivIVA. Bacillus subtilis MinD has two amphipathic α-helices rich in basic amino acid residues at its C-terminus; one of these, named MTS1 here, is the counterpart of the membrane targeting sequence (MTS) in Escherichia coli MinD while the other, named MTS-like sequence (MTSL), is the nearest helix to MTS1. These amphipathic helices were located independently at nascent septal membranes in cells lacking MinJ or DivIVA, whereas elimination of the helices from the wild type protein reduced its localization considerably. MinD variants with altered MTS1 and MTSL, in which basic amino acid residues were replaced with proline or acidic residues, were not located at nascent septal membranes, indicating that the binding to the nascent septal membranes requires basic residues and a helical structure. The septal localization of MTSL, but not of MTS1, was dependent on host cell MinD. These results suggest that MinD is targeted to nascent septal membranes via its C-terminal amphipathic α-helices in B. subtilis cells lacking MinJ or DivIVA. Moreover, the diffuse distribution of MinD lacking both MTSs suggests that only a small fraction of MinD depends on MinJ for its localization to nascent septal membranes.

  7. First Delayed Resection Findings After Irreversible Electroporation (IRE) of Human Localised Renal Cell Carcinoma (RCC) in the IRENE Pilot Phase 2a Trial

    Energy Technology Data Exchange (ETDEWEB)

    Wendler, Johann Jakob, E-mail: johann.wendler@med.ovgu.de [Otto von Guericke University of Magdeburg, Department of Urology, University Hospital (Germany); Ricke, Jens, E-mail: jens.Ricke@med.ovgu.de; Pech, Maciej, E-mail: macej.pech@med.ovgu.de; Fischbach, Frank, E-mail: frank.fischbach@med.ovgu.de; Jürgens, Julian, E-mail: julian.juergens@med.ovgu.de [University of Magdeburg, Department of Radiology (Germany); Siedentopf, Sandra, E-mail: sandra.siedentopf@med.ovgu.de; Roessner, Albert, E-mail: albert.roessner@med.ovgu.de [University of Magdeburg, Institute of Pathology (Germany); Porsch, Markus, E-mail: markus.porsch@med.ovgu.de; Baumunk, Daniel, E-mail: daniel.baumunk@med.ovgu.de; Schostak, Martin, E-mail: martin.schostak@med.ovgu.de [Otto von Guericke University of Magdeburg, Department of Urology, University Hospital (Germany); Köllermann, Jens, E-mail: jens.koellermann@sana.de [Sana Klinikum Offenbach Am Main, Institute of Pathology (Germany); Liehr, Uwe-Bernd, E-mail: uwe-bernd.liehr@med.ovgu.de [Otto von Guericke University of Magdeburg, Department of Urology, University Hospital (Germany)

    2016-02-15

    IntroductionIt is postulated that focal IRE affords complete ablation of soft-tissue tumours while protecting the healthy peritumoral tissue. Therefore, IRE may be an interesting option for minimally invasive, kidney-tissue-sparing, non-thermal ablation of renal tumours.AimWith this current pilot study (“IRENE trial”), we present the first detailed histopathological data of IRE of human RCC followed by delayed tumour resection. The aim of this interim analysis of the first three patients was to investigate the ablation efficiency of percutaneous image-guided focal IRE in RCC, to assess whether a complete ablation of T1a RCC and tissue preservation with the NanoKnife system is possible and to decide whether the ablation parameters need to be altered.MethodsFollowing resection 4 weeks after percutaneous IRE, the success of ablation and detailed histopathological description were used to check the ablation parameters.ResultsThe IRE led to a high degree of damage to the renal tumours (1 central, 2 peripheral; size range 15–17 mm). The postulated homogeneous, isomorphic damage was only partly confirmed. We found a zonal structuring of the ablation zone, negative margins and, enclosed within the ablation zone, very small tumour residues of unclear malignancy.ConclusionAccording to these initial, preliminary study results of the first three renal cases, a new zonal distribution of IRE damage was described and the curative intended, renal saving focal ablation of localised RCC below <3 cm by percutaneous IRE by the NanoKnife system appears to be possible, but needs further, systematic evaluation for this treatment method and treatment protocol.

  8. Increased expression of IRE1α and stress-related signal transduction proteins in ischemia-reperfusion injured retina

    Directory of Open Access Journals (Sweden)

    Natsuyo Hata

    2008-08-01

    Full Text Available Natsuyo Hata1, Toshiyuki Oshitari1,2, Akiko Yokoyama1,3, Yoshinori Mitamura1, Shuichi Yamamoto11Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chuo-ku, Chiba, Japan; 2Department of Ophthalmology, Kimitsu Central Hospital, Kisarazu City, Chiba, Japan; 3Department of Ophthalmology, Inoue Memorial Hospital, Chuo-ku, Chiba, JapanAbstract: The purpose of this study was to determine whether the expression of ER stress-related factors IRE1α, apoptosis signal-regulating kinase 1 (ASK1, SAPK/ERK kinase 1 (SEK1 and c-Jun N-terminal kinase (JNK is associated with the damaged retinal neurons induced by ischemia-reperfusion injury. After 60 minutes of ischemia, the rat retinas were reperfused, and retinas were isolated and fixed after 6, 9, 12, 18, and 24 hours, and 2, 5, and 9 days of reperfusion. Cryosections were immunostained with Fluoro-Jade B, a degenerating neuron marker to label degenerating neurons. Semi-quantitative analysis of the expression of IRE1α, ASK1, SEK1, and JNK were performed in both control and ischemic retinas. In ischemic retinas, the intensities of IRE1α immunoreactivity in the ganglion cell layer (GCL were significantly higher than in the control retinas. In ischemic retinas, the numbers of SEK1-, ASK1-, and JNK-positive cells were significantly increased in the GCL compared to those in the control retinas. In addition, the cells that were positive for SEK1-, ASK1-, and JNK were also positive for Fluoro-Jade B-positive cells. These results indicate that the increased expression of ER stress-related factors was, in part, associated with the retinal neuronal abnormalities after ischemia-reperfusion injury in rat retinas.Keywords: endoplasmic reticulum, IRE1α, apoptosis signal-regulating kinase 1, SAPK/ERK kinase 1, c-Jun N-terminal kinase, Fluoro-Jade B, ischemia-reperfusion injury

  9. Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

    Science.gov (United States)

    Sepulveda, Denisse; Rojas-Rivera, Diego; Rodríguez, Diego A; Groenendyk, Jody; Köhler, Andres; Lebeaupin, Cynthia; Ito, Shinya; Urra, Hery; Carreras-Sureda, Amado; Hazari, Younis; Vasseur-Cognet, Mireille; Ali, Maruf M U; Chevet, Eric; Campos, Gisela; Godoy, Patricio; Vaisar, Tomas; Bailly-Maitre, Béatrice; Nagata, Kazuhiro; Michalak, Marek; Sierralta, Jimena; Hetz, Claudio

    2018-01-18

    Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Somatostatin-IRES-Cre Mice: Between Knockout and Wild-Type?

    Science.gov (United States)

    Viollet, Cécile; Simon, Axelle; Tolle, Virginie; Labarthe, Alexandra; Grouselle, Dominique; Loe-Mie, Yann; Simonneau, Michel; Martel, Guillaume; Epelbaum, Jacques

    2017-01-01

    The neuropeptide somatostatin (SOM) is widely expressed in rodent brain and somatostatin-IRES-Cre (SOM-cre) mouse strains are increasingly used to unravel the physiology of SOM-containing neurons. However, while knock-in targeting strategy greatly improves Cre-Lox system accuracy, recent reports have shown that genomic insertion of Cre construct per se can markedly affect physiological function. We show that Cre transgene insertion into the 3'UTR of the somatostatin gene leads to the selective and massive depletion of endogenous SOM in all tested brain regions. It also strongly impacts SOM-related neuroendocrine responses in a similar manner to what has been reported for SST KO mice: increased corticosterone levels after 30-min restraint stress, decreased amplitude and regularity of ultradian growth hormone secretory patterns accompanied by changes in sexually dimorphic liver gene expression ( serpina1, Cyp2b9, Cyp2a4, Cyp2d9, and Cyp7b1 ). In addition to demonstrating the need for examination of the consequences of Cre transgenesis, these results also reveal how this SOM-cre strain may be a useful tool in studying the functional consequences of moderate to low SOM levels as reported in neurological and psychiatric disorders.

  11. Undergraduate Student Involvement in International Research - The IRES Program at MAX-lab, Sweden

    Science.gov (United States)

    Briscoe, William; O'Rielly, Grant; Fissum, Kevin

    2014-03-01

    Undergraduate students associated with The George Washington University and UMass Dartmouth have had the opportunity to participate in nuclear physics research as a part of the PIONS@MAXLAB Collaboration performing experiments at MAX-lab at Lund University in Sweden. This project has supported thirteen undergraduate students during 2009 - 2011. The student researchers are involved with all aspects of the experiments performed at the laboratory, from set-up to analysis and presentation at national conferences. These experiments investigate the dynamics responsible for the internal structure of the nucleon through the study of pion photoproduction off the nucleon and high-energy Compton scattering. Along with the US and Swedish project leaders, members of the collaboration (from four different countries) have contributed to the training and mentoring of these students. This program provides students with international research experiences that prepare them to operate successfully in a global environment and encourages them to stay in areas of science, technology, engineering and math (STEM) that are crucial for our modern, technology-dependent society. We will present the history, goals and outcomes in both physics results and student success that have come from this program. This work supported by NSF OISE/IRES award 0553467.

  12. Non-coding RNA in Deinococcus radiodurans

    International Nuclear Information System (INIS)

    Chen Zhongzhong; Wang Liangyan; Lin Jun; Tian Bing; Hua Yuejin

    2006-01-01

    Researches on DNA damage and repair pathways of Deinococcus radiodurans show its extreme resistance to ionizing radiation, ultraviolet radiation and reactive oxygen species. Non-coding (ncRNA) RNAs are involved in a variety of processes such as transcriptional regulations, RNA processing and modification, mRNA translation, protein transportation and stability. The conserved secondary structures of intergenic regions of Deinococcus radiodurans R1 were predicted using Stochastic Context Free Grammar (SCFG) scan strategy. Results showed that 28 ncRNA families were present in the non-coding regions of the genome of Deinococcus radiodurans R1. Among these families, IRE is the largest family, followed by Histone3, tRNA, SECIS. DicF, ctRNA-pGA1 and tmRNA are one discovered in bacteria. Results from the comparison with other organisms showed that these ncRNA can be applied to the study of biological function of Deinococcus radiodurans and supply reference for the further study of DNA damage and repair mechanisms of this bacterium. (authors)

  13. MRI and contrast-enhanced ultrasound imaging for evaluation of focal irreversible electroporation treatment: results from a phase I-II study in patients undergoing IRE followed by radical prostatectomy

    International Nuclear Information System (INIS)

    Bos, Willemien van den; Bruin, D.M. de; Randen, A. van; Engelbrecht, M.R.W.; Postema, A.W.; Muller, B.G.; Zondervan, P.J.; Laguna Pes, M.P.; Reijke, T.M. de; Rosette, J.J.M.C.H. de la; Varkarakis, I.M.; Skolarikos, A.; Savci-Heijink, C.D.; Jurhill, R.R.; Wijkstra, H.

    2016-01-01

    Irreversible electroporation (IRE) is an ablative therapy with a low side-effect profile in prostate cancer. The objective was: 1) To compare the volumetric IRE ablation zone on grey-scale transrectal ultrasound (TRUS), contrast-enhanced ultrasound (CEUS) and multiparametric MRI (mpMRI) with histopathology findings; 2) To determine a reliable imaging modality to visualize the IRE ablation effects accurately. A prospective phase I-II study was performed in 16 patients scheduled for radical prostatectomy (RP). IRE of the prostate was performed 4 weeks before RP. Prior to, and 4 weeks after the IRE treatment, imaging was performed by TRUS, CEUS, and mpMRI. 3D-analysis of the ablation volumes on imaging and on H and E-stained whole-mount sections was performed. The volumes were compared and the correlation was calculated. Evaluation of the imaging demonstrated that with T2-weighted MRI, dynamic contrast enhanced (DCE) MRI, and CEUS, effects of IRE are visible. T2MRI and CEUS closely match the volumes on histopathology (Pearson correlation r = 0.88 resp. 0.80). However, IRE is not visible with TRUS. mpMRI and CEUS are appropriate for assessing IRE effects and are the most feasible imaging modalities to visualize IRE ablation zone. The imaging is concordant with results of histopathological examination. (orig.)

  14. Hypoxic regulation of the expression of genes encoded estrogen related proteins in U87 glioma cells: eff ect of IRE1 inhibition.

    Science.gov (United States)

    Minchenko, D O; Riabovol, O O; Ratushna, O O; Minchenko, O H

    2017-01-01

    The aim of the present study was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoded estrogen related proteins (NRIP1/RIP140, TRIM16/EBBP, ESRRA/NR3B1, FAM162A/E2IG5, PGRMC2/PMBP, and SLC39A6/LIV-1) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of glioma cells proliferation. The expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by a quantitative polymerase chain reaction. Inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 signaling enzyme function up-regulates the expression of EBBP, E2IG5, PGRMC2, and SLC39A6 genes is in U87 glioma cells in comparison with the control glioma cells, with more significant changes for E2IG5 and PGRMC2 genes. At the same time, the expression of NRIP1 and ESRRA genes is strongly down-regulated in glioma cells upon inhibition of IRE1. We also showed that hypoxia increases the expression of E2IG5, PGRMC2, and EBBP genes and decreases NRIP1 and ESRRA genes expression in control glioma cells. Furthermore, the inhibition of IRE1 in U87 glioma cells decreases the eff ect of hypoxia on the expression of E2IG5 and PGRMC2 genes, eliminates hypoxic regulation of NRIP1 gene, and enhances the sensitivity of ESRRA gene to hypoxic condition. Furthermore, the expression of SLC39A6 gene is resistant to hypoxia in both the glioma cells with and without IRE1 signaling enzyme function. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells in gene specific manner and these changes

  15. Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing

    International Nuclear Information System (INIS)

    Ri, M; Tashiro, E; Oikawa, D; Shinjo, S; Tokuda, M; Yokouchi, Y; Narita, T; Masaki, A; Ito, A; Ding, J; Kusumoto, S; Ishida, T; Komatsu, H; Shiotsu, Y; Ueda, R; Iwawaki, T; Imoto, M; Iida, S

    2012-01-01

    The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy

  16. Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors.

    Science.gov (United States)

    López-Lastra, M; Gabus, C; Darlix, J L

    1997-11-01

    The murine leukemia virus (MLV)-related type C viruses constitute a major class of retroviruses that includes numerous endogenous and exogenous mammalian viruses and the related avian spleen necrosis virus (SNV). The MLV-related viruses possess a long and multifunctional 5' untranslated leader involved in key steps of the viral life cycle--splicing, translation, RNA dimerization, encapsidation, and reverse transcription. Recent studies have shown that the 5' leader of Friend murine leukemia virus and Moloney murine leukemia virus can direct cap independent translation of gag precursor proteins (Berlioz et al., 1995; Vagner et al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new internal ribosome entry segment (IRES) of retroviral origin. Here we describe an IRES element within the 5' leader of avian reticuloendotheliosis virus type A (REV-A) genomic RNA. Data show that the REV-A 5' IRES element maps downstream of the packaging/dimerization (E/DLS) sequence (Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES sequence appears to be within a 129 nt fragment (nucleotides 452-580) of the 5' leader, immediately upstream of the gag AUG codon. The REV-A IRES has been successfully utilized in the construction of novel high titer MLV-based retroviral vectors, containing one or more IRES elements of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene therapy, are also of interest as a model system of internal translation initiation and its possible regulation during development, cancer, or virus infection.

  17. An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype

    International Nuclear Information System (INIS)

    McKnight, Kevin L.; Sandefur, Stephanie; Phipps, Krista M.; Heinz, Beverly A.

    2003-01-01

    The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs

  18. Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

    Science.gov (United States)

    Kodama, Rika; Byun, Sanguine; Yoon, Kyoung Wan; Hiraki, Masatsugu; Mandinova, Anna; Lee, Sam W.

    2015-01-01

    Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53. PMID:26254280

  19. The Italian forest sites of FunDivEUROPE: a new FP7 project on the functional significance of forest biodiversity in Europe

    Directory of Open Access Journals (Sweden)

    Bussotti F

    2012-12-01

    Full Text Available The Italian forest sites of FunDivEUROPE: a new FP7 project on the functional significance of forest biodiversity in Europe. FunDivEUROPE is a new project aiming at a deeper understanding of the role of forest diversity on ecosystem functions and service provisioning for society. This project combines three scientific platforms: experimental, exploratory and inventory. The exploratory platform is based on the observation of a broad range of properties, traits and ecological processes on a network of ca. 240 natural forest sites representing a gradient of tree species diversity in six focal regions of Europe (Spain, Italy, Germany, Poland, Finland and Romania. The Italian sites are located on the hills of central and Southern Tuscany and represent the category “thermophilous deciduous forest”. Almost one year of fieldwork was needed to select and characterize 36 plots measuring 30 x 30 m. Selection was based on criteria concerning tree mixtures and richness, structural parameters and main environmental variables. The main features of these sites are synthetically presented in this paper together with a short description of the project structure and scope. The aim is also to enhance dissemination of the potential implications for a sustainable forest management in Italy.

  20. BECCS Market Launch Strategy Aiming to Help Ensure Reliable Grid Power at High Penetrations of IRE (Intermittent Renewable Electricity)

    Science.gov (United States)

    WIlliams, R. H.

    2017-12-01

    Despite its recognized importance for carbon (C)-mitigation, progress in advancing biomass energy with CO2 capture and sequestration (BECCS) has been slow. A BECCS market launch strategy based on technologies ready for commercial-scale demonstration is discussed—based on co-gasification of coal and biomass to make H2 with CCS. H2 so produced would be a key element of a H2 balancing capacity (H2-BC) strategy for ensuring reliable grid power at high IRE penetrations. High grid penetrations of IRE must be complemented by fast-ramping balancing (backup and/or storage) capacity (BC) to ensure reliable grid power. BC provided now by natural gas-fired gas turbine combined cycle and combustion turbine units would eventually have to be decarbonized to realize C-mitigation goals, via CCS or other means. Capital-intensive CCS energy systems require baseload operation to realize favourable economics, but at high IRE penetrations, BC plants must be operated at low capacity factors. A H2-BC strategy is a promising way to address this challenge. The elements of a H2-BC system are: (a) H2 production from carbonaceous feedstocks in baseload plants with CCS; (b) H2 consumption in fast-ramping BC units that operate at low capacity factors; (c) Buffer underground H2 storage to enable decoupling baseload H2 production from highly variable H2 consumption by BC units. The concept is likely to "work" because underground H2 storage is expected to be inexpensive. A H2 production analysis is presented for a negative GHG-emitting H2-BC system based on cogasification of corn stover and coal, with captured CO2 used for enhanced oil recovery. The technical readiness of each system component is discussed, and preliminary insights are offered as to the conditions under which the corresponding H2-BC system might compete with natural gas in providing backup for IRE on US electric grids. Public policy to help advance this strategy might be forthcoming, because 2 US Senate bills with broad

  1. Ground Reaction Force and Mechanical Differences Between the Interim Resistive Exercise Device (iRED) and Smith Machine While Performing a Squat

    Science.gov (United States)

    Amonette, William E.; Bentley, Jason R.; Lee, Stuart M. C.; Loehr, James A.; Schneider, Suzanne

    2004-01-01

    Musculoskeletal unloading in microgravity has been shown to induce losses in bone mineral density, muscle cross-sectional area, and muscle strength. Currently, an Interim Resistive Exercise Device (iRED) is being flown on board the ISS to help counteract these losses. Free weight training has shown successful positive musculoskeletal adaptations. In biomechanical research, ground reaction forces (GRF) trajectories are used to define differences between exercise devices. The purpose of this evaluation is to quantify the differences in GRF between the iRED and free weight exercise performed on a Smith machine during a squat. Due to the differences in resistance properties, inertial loading and load application to the body between the two devices, we hypothesize that subjects using iRED will produce GRF that are significantly different from the Smith machine. There will be differences in bar/harness range of motion and the time when peak GRF occurred in the ROMbar. Three male subjects performed three sets of ten squats on the iRED and on the Smith Machine on two separate days at a 2-second cadence. Statistically significant differences were found between the two devices in all measured GRF variables. Average Fz and Fx during the Smith machine squat were significantly higher than iRED. Average Fy (16.82 plus or minus.23; p less than .043) was significantly lower during the Smith machine squat. The mean descent/ascent ratio of the magnitude of the resultant force vector of all three axes for the Smith machine and iRED was 0.95 and 0.72, respectively. Also, the point at which maximum Fz occurred in the range of motion (Dzpeak) was at different locations with the two devices.

  2. Yip1A, a Novel Host Factor for the Activation of the IRE1 Pathway of the Unfolded Protein Response during Brucella Infection

    Science.gov (United States)

    Taguchi, Yuki; Imaoka, Koichi; Kataoka, Michiyo; Uda, Akihiko; Nakatsu, Daiki; Horii-Okazaki, Sakuya; Kunishige, Rina; Kano, Fumi; Murata, Masayuki

    2015-01-01

    Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments.  On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation. PMID:25742138

  3. Read-Split-Run: an improved bioinformatics pipeline for identification of genome-wide non-canonical spliced regions using RNA-Seq data.

    Science.gov (United States)

    Bai, Yongsheng; Kinne, Jeff; Donham, Brandon; Jiang, Feng; Ding, Lizhong; Hassler, Justin R; Kaufman, Randal J

    2016-08-22

    Most existing tools for detecting next-generation sequencing-based splicing events focus on generic splicing events. Consequently, special types of non-canonical splicing events of short mRNA regions (IRE1α targeted) have not yet been thoroughly addressed at a genome-wide level using bioinformatics approaches in conjunction with next-generation technologies. During endoplasmic reticulum (ER) stress, the gene encoding the RNase Ire1α is known to splice out a short 26 nt region from the mRNA of the transcription factor Xbp1 non-canonically within the cytosol. This causes an open reading frame-shift that induces expression of many downstream genes in reaction to ER stress as part of the unfolded protein response (UPR). We previously published an algorithm termed "Read-Split-Walk" (RSW) to identify non-canonical splicing regions using RNA-Seq data and applied it to ER stress-induced Ire1α heterozygote and knockout mouse embryonic fibroblast cell lines. In this study, we have developed an improved algorithm "Read-Split-Run" (RSR) for detecting genome-wide Ire1α-targeted genes with non-canonical spliced regions at a faster speed. We applied the RSR algorithm using different combinations of several parameters to the previously RSW tested mouse embryonic fibroblast cells (MEF) and the human Encyclopedia of DNA Elements (ENCODE) RNA-Seq data. We also compared the performance of RSR with two other alternative splicing events identification tools (TopHat (Trapnell et al., Bioinformatics 25:1105-1111, 2009) and Alt Event Finder (Zhou et al., BMC Genomics 13:S10, 2012)) utilizing the context of the spliced Xbp1 mRNA as a positive control in the data sets we identified it to be the top cleavage target present in Ire1α (+/-) but absent in Ire1α (-/-) MEF samples and this comparison was also extended to human ENCODE RNA-Seq data. Proof of principle came in our results by the fact that the 26 nt non-conventional splice site in Xbp1 was detected as the top hit by our new RSR

  4. The long noncoding RNA RNCR2 directs mouse retinal cell specification

    Directory of Open Access Journals (Sweden)

    Blackshaw Seth

    2010-05-01

    Full Text Available Abstract Background Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation. Results We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation. Conclusion These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.

  5. 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

  6. A close connection between the PERK and IRE arms of the UPR and the transcriptional regulation of autophagy.

    Science.gov (United States)

    Deegan, Shane; Koryga, Izabela; Glynn, Sharon A; Gupta, Sanjeev; Gorman, Adrienne M; Samali, Afshin

    2015-01-02

    Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Accidental release of iodine 131 by the IRE of the Fleurus site: return on experience by the Belgian safety authority

    International Nuclear Information System (INIS)

    Vandecasteele, C.M.; Sonck, M.; Degueldre, D.

    2010-01-01

    After a presentation of the activities of the IRE, the Belgian National Institute of Radio-elements, i.e. the production of radionuclides used in nuclear medicine, this report describes the process and chemical reaction which caused an accidental release of iodine 131. It analyzes the causes of this incident, and how the incident has been managed by the Belgian safety authority. It discusses the first assessment of radiological consequences, describes how the incident has been managed at the federal level, and how population and media have been informed. It discusses the actual radiological consequences through measurements performed on grass and vegetables (graphs and maps indicate contamination levels and contaminated areas), and through the assessment of exposure of adults and children by different ways. Lessons learned are then discussed

  8. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  9. O envolvimento parental após a separação/divórcio Parental involvement after separation/divorce

    Directory of Open Access Journals (Sweden)

    Luciana Suárez Grzybowski

    2010-01-01

    Full Text Available Apoiados no modelo ecológico-contextual e no conceito de envolvimento parental, este artigo se propõe a discutir as práticas parentais de pais e mães separados/divorciados com seus filhos. Para tanto, foram pesquisados 234 sujeitos (117 pais/117 mães com filhos em idade escolar, que responderam o Inventário de Práticas Parentais. Tal instrumento avaliou o envolvimento parental em 5 áreas: envolvimento afetivo, envolvimento didático, envolvimento social, envolvimento disciplinar e responsabilidade. Os resultados evidenciam maior envolvimento materno do que paterno com os filhos após o divórcio, tanto direto (cuidados, interação quanto indireto (monitoramento, preocupação. A coabitação com a mãe mostrou ser uma variável significativa associada ao maior envolvimento dela em atividades no espaço privado/doméstico, enquanto os pais tiveram maior envolvimento no espaço público/social. Características contextuais (coabitação, freqüência de visitas e características dos pais (ocupação, escolaridade, questões afetivo-conjugais mostraram-se fortemente associadas ao envolvimento parental após o divórcio.Based on the ecological-contextual model and on the concept of parental involvement, this article proposes to discuss the parental practices of separated/divorced fathers and mothers towards their children. In order to do so, 234 subjects (117 fathers/117 mothers with children at school were interviewed via the Parental Practices Inventory. Such instrument evaluated the parental involvement in 5 areas: affective involvement, didactic involvement, social involvement, disciplinary involvement and, responsibility. The results showed greater maternal involvement with children after the divorce: direct (care, interaction and indirect (monitoring, preoccupation. The cohabitation with the mother revealed itself as a significant variable associated to her greater involvement with activities in the private/domestic environment while

  10. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

  11. Cis-regulatory RNA elements that regulate specialized ribosome activity.

    Science.gov (United States)

    Xue, Shifeng; Barna, Maria

    2015-01-01

    Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5'UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution.

  12. Structural Analysis of PTM Hotspots (SAPH-ire)--A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families.

    Science.gov (United States)

    Dewhurst, Henry M; Choudhury, Shilpa; Torres, Matthew P

    2015-08-01

    Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)--a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits--conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit-N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data. © 2015 by The American

  13. Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families*

    Science.gov (United States)

    Dewhurst, Henry M.; Choudhury, Shilpa; Torres, Matthew P.

    2015-01-01

    Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data. PMID:26070665

  14. Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Response Pathway Genes skn-1, ire-1, and xbp-1

    Directory of Open Access Journals (Sweden)

    Karla A. Mark

    2016-10-01

    Full Text Available Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer’s disease, Parkinson’s disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3 induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human β-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency.

  15. High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71.

    Science.gov (United States)

    Levengood, Jeffrey D; Tolbert, Michele; Li, Mei-Ling; Tolbert, Blanton S

    2013-07-01

    Human Enterovirus 71 (EV71) is an emerging pathogen of infectious disease and a serious threat to public health. Currently, there are no antivirals or vaccines to slow down or prevent EV71 infections, thus underscoring the urgency to better understand mechanisms of host-enterovirus interactions. EV71 uses a type I internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit via a pathway that requires the cytoplasmic localization of hnRNP A1, which acts as an IRES trans-activating factor. The mechanism of how hnRNP A1 trans activates EV71 RNA translation is unknown, however. Here, we report that the UP1 domain of hnRNP A1 interacts specifically with stem loop II (SLII) of the IRES, via a thermodynamically well-defined biphasic transition that involves conserved bulge 5'-AYAGY-3' and hairpin 5'-RY(U/A)CCA-3' loops. Calorimetric titrations of wild-type and mutant SLII constructs reveal these structural elements are essential to form a high-affinity UP1-SLII complex. Mutations that alter the bulge and hairpin primary or secondary structures abrogate the biphasic transition and destabilize the complex. Notably, mutations within the bulge that destabilize the complex correlate with a large reduction in IRES-dependent translational activity and impair EV71 replication. Taken together, this study shows that a conserved SLII structure is necessary to form a functional hnRNP A1-IRES complex, suggesting that small molecules that target this stem loop may have novel antiviral properties.

  16. DIvERTICULE DE ZENKER

    African Journals Online (AJOL)

    origine de complications respiratoires aigues (détresse respiratoire aigue, pneumopathie d'inha- lation) ou chronique (bronchite chronique, asthme) qui surviennent dans 20 % des cas [8]. L'examen clinique est souvent normal. Cependant une.

  17. Adaptação ao divórcio e relações coparentais: contributos da teoria da vinculação Adjustment to divorce and co-parental relations: contributions from the theory of attachment

    Directory of Open Access Journals (Sweden)

    Diogo Lamela

    2010-01-01

    Full Text Available Neste artigo propõe-se uma contribuição da teoria da vinculação na compreensão dos processos de adaptação dos adultos ao seu divórcio e como a desvinculação ao ex-cônjuge interfere na coparentalidade pós-divórcio. Este artigo formula duas hipóteses teóricas. A primeira hipótese afirma que o divórcio, enquanto processo relacional, deve ser lido como um momento de perda que germina reacções psicológicas similares às experienciadas pelos viúvos, tal como descreve Bowlby no modelo de perda da figura de vinculação, estando dependente dos estilos de vinculação dos adultos. A segunda hipótese sustenta que a coparentalidade pós-divórcio é predita pelos estilos de vinculação e pela qualidade da reorganização da vinculação dos pais. Finalmente, uma integração teórica é apresentada, operacionalizada numa proposta de investigação futura neste domínio.In this article, it is proposed the contribution of the attachment theory for understanding adults' adjustment processes to their divorce and how detachment to ex-spouse can infer in co-parenting relationships after marital dissolution. This article makes two theoretical assumptions that focus on two dimensions. The first hypothesis states that the divorce, while a relational process, should be read as a moment of loss that germinates similar psychological reactions to those experienced by widows. Bowlby describes it in his model of loss of the attachment figure as dependent on attachment styles of divorced adults. The second hypothesis argues that the post-divorce co-parenting relationships are predicted by the attachment styles and by the quality of parents' attachment reorganization. At the end, a theoretical integration is built, based on a proposal for future research in this area.

  18. Assessment of the dentoskeletal changes following the use of twin-block functional appliance and inclined anterior bite plan in the patients with class II malocclusion div 1 in 11-14 years old

    Directory of Open Access Journals (Sweden)

    Allahyar Geramy

    2015-12-01

    Full Text Available Background and Aims: Cl II malocclusion is one of the most common abnormalities in human societies and using a simple, affordable and accessible treatment that can be provided by general practitioners or specialists, to prevent future malocclusion complications is vital. Cl II malocclusion treatment using functional appliances such as twin block and anterior inclined bite plan have less cost, side effects and complications compared to fixed orthodontic or orthosurgery. Until now, the dentoskeletal changes resulting from the application of twin block and anterior inclined bite plan in patients with Class II malocclusion has not been evaluated. The objective of the present study was to assess the dentoskeletal changes following the use of twin-block functional appliance and inclined anterior bite plan in the patients with class II malocclusion div 1 during mixed dentition. Materials and Methods: In this retrospective cohort trial, 60 patients with the definitive diagnosis of Class II div. 1 malocclusion having ANB>2 and FMA angel between 20 and 30 and without any previous treatment or syndrome who were treated with twin-block or inclined anterior bite plan appliance were selected and their lateral cephalometries were traced before and after treatment. Selected distance and angular landmarks were measured on the cephalograms with the good reliability (ICC=0.953 and the changes occurred in the landmarks were statistically analyzed using Student t test. Results: Due to the increased mandibular growth, most of the landmarks experienced significant changes following the treatment with twin-block and inclined bite plan (P0.05, however, SNA (P=0.04, overjet (P=0.007 and wits appraisal (P=0.004 changed differently after using the appliances. Conclusion: Despite with most similarities of both twin-block and anterior inclined bite plan to correct class II div. 1 malocclusion, Due to the advantages of anterior inclined bite plan such as less size, good

  19. ZmbZIP60 mRNA is spliced in maize in response to ER stress

    Directory of Open Access Journals (Sweden)

    Li Yanjie

    2012-03-01

    Full Text Available Abstract Background Adverse environmental conditions produce ER stress and elicit the unfolded protein response (UPR in plants. Plants are reported to have two "arms" of the ER stress signaling pathway-one arm involving membrane-bound transcription factors and the other involving a membrane-associated RNA splicing factor, IRE1. IRE1 in yeast to mammals recognizes a conserved twin loop structure in the target RNA. Results A segment of the mRNA encoding ZmbZIP60 in maize can be folded into a twin loop structure, and in response to ER stress this mRNA is spliced, excising a 20b intron. Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus. Splicing of ZmbZIP60 can be elicited in maize seedlings by ER stress agents such as dithiothreitol (DTT or tunicamycin (TM or by heat treatment. Younger, rather than older seedlings display a more robust splicing response as do younger parts of leaf, along a developmental gradient in a leaf. The molecular signature of an ER stress response in plants includes the upregulation of Binding Protein (BIP genes. Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb. Conclusions The splicing of ZmbZIP60 mRNA is an indicator of ER stress in maize seedlings resulting from adverse environmental conditions such as heat stress. ZmbZIP60 mRNA splicing in maize leads predictively to the formation of active bZIP transcription factor targeted to the nucleus to upregulate stress response genes. Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

  20. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

    Science.gov (United States)

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

    2017-04-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  1. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    Science.gov (United States)

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

    2017-01-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

  2. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    .9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...

  3. Expression of heterologous genes from an IRES translational cassette in replication-competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, T.; Duch, M.; Carrasco, M.L.

    1999-01-01

    of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  4. Isikult vabaduse võtmine põhjendusel, et ta on psüühikahäire tõttu endale või teistele ohtlik / Sten Lind, Katrin Eino

    Index Scriptorium Estoniae

    Lind, Sten, 1979-

    2014-01-01

    Psüühikahäire määratlemine ning isiku ohtlikkus psühhiaatrilise abi seaduse § 11, sotsiaalhoolekande seaduse § 19 ja karistusseadustiku § 86 kontekstis. Riigikohtu lahenditest 3-2-1-81-07 ja 3-2-1-155-13

  5. Papel da via de sinalização mediada por Ire1 e Xbp1 na diferenciação de fotoreceptores em Drosophila

    OpenAIRE

    Coelho, Dina Raquel da Silva, 1984-

    2009-01-01

    Tese de mestrado. Biologia (Biologia Molecular Humana). Universidade de Lisboa, Faculdade de Ciências, 2009 O mecanismo de Unfolded Protein Response (UPR) protege as células do stress no retículo endoplasmático (RE) causado pela acumulação de proteínas misfolded no seu lúmen. Este mecanismo leva à activação de um conjunto de vias de sinalização que aumenta a capacidade de folding de proteínas no RE. No presente trabalho, analisámos o papel da via do UPR mediada por Ire1 e Xbp1 no desenvolv...

  6. An approach to the construction of tailor-made amphiphilic peptides that strongly and selectively bind to hairpin RNA targets.

    Science.gov (United States)

    Lee, Su Jin; Hyun, Soonsil; Kieft, Jeffrey S; Yu, Jaehoon

    2009-02-18

    The hairpin RNA motif is one of the most frequently observed secondary structures and is often targeted by therapeutic agents. An amphiphilic peptide with seven lysine and eight leucine residues and its derivatives were designed for use as ligands against RNA hairpin motifs. We hypothesized that variations in both the hydrophobic leucine-rich and hydrophilic lysine-rich spheres of these amphiphilic peptides would create extra attractive interactions with hairpin RNA targets. A series of alanine-scanned peptides were probed to identify the most influential lysine residues in the hydrophilic sphere. The binding affinities of these modified peptides with several hairpins, such as RRE, TAR from HIV, a short hairpin from IRES of HCV, and a hairpin from the 16S A-site stem from rRNA, were determined. Since the hairpin from IRES of HCV was the most susceptible to the initial series of alanine-scanned peptides, studies investigating how further variations in the peptides effect binding employed the IRES hairpin. Next, the important Lys residues were substituted by shorter chain amines, such as ornithine, to place the peptide deeper into the hairpin groove. In a few cases, a 70-fold improved binding was observed for peptides that contained the specifically located shorter amine side chains. To further explore changes in binding affinities brought about by alterations in the hydrophobic sphere, tryptophan residues were introduced in place of leucine. A few peptides with tryptophan in specific positions also displayed 70-fold improved binding affinities. Finally, double mutant peptides incorporating both specifically located shorter amine side chains in the hydrophilic region and tryptophan residues in the hydrophobic region were synthesized. The binding affinities of peptides containing the simple double modification were observed to be 80 times lower, and their binding specificities were increased 40-fold. The results of this effort provide important information about

  7. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

    Science.gov (United States)

    Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

    2017-01-01

    Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

  8. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

    Directory of Open Access Journals (Sweden)

    Peng Li

    2017-09-01

    Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

  9. Extracellular RNA Communication (ExRNA)

    Data.gov (United States)

    Federal Laboratory Consortium — Until recently, scientists believed RNA worked mostly inside the cell that produced it. Some types of RNA help translate genes into proteins that are necessary for...

  10. An RNA secondary structure bias for non-homologous reverse transcriptase-mediated deletions in vivo

    DEFF Research Database (Denmark)

    Duch, Mogens; Carrasco, Maria L; Jespersen, Thomas

    2004-01-01

    Murine leukemia viruses harboring an internal ribosome entry site (IRES)-directed translational cassette are able to replicate, but undergo loss of heterologous sequences upon continued passage. While complete loss of heterologous sequences is favored when these are flanked by a direct repeat......, deletion mutants with junction sites within the heterologous cassette may also be retrieved, in particular from vectors without flanking repeats. Such deletion mutants were here used to investigate determinants of reverse transcriptase-mediated non-homologous recombination. Based upon previous structural...... result from template switching during first-strand cDNA synthesis and that the choice of acceptor sites for non-homologous recombination are guided by non-paired regions. Our results may have implications for recombination events taking place within structured regions of retroviral RNA genomes...

  11. Micro RNA, A Review: Pharmacogenomic drug targets for complex diseases

    Directory of Open Access Journals (Sweden)

    Sandhya Bawa

    2010-01-01

    Full Text Available <div>

    Micro RNAs (miRNAs are non-coding RNAs that can regulate gene expression to target several mRNAs in a gene regulatory network. MiRNA related Single Nucleotide Polymorphisms (S.N.P.s represent a newly identified type of genetic variability that can be of influence to the risk of certain human diseases and also affect how drugs can be activated and metabolized by patients. This will help in personalized medicines which are used for administrating the correct dosage of drug and drug efficacy. miRNA deregulated expression has been extensively described in a variety of diseases such as Cancer, Obesity , Diabetes, Schizophrenia and control and self renewal of stem cells. MiRNA can function as oncogenes and/or tumor suppressors. MiRNAs may act as key regulators of processes as diverse as early development, cell proliferation and cell death, apoptosis and fat metabolism and cell differentiation .miRNA expression have shown their role in brain development chronic lymphocytic leukemia, colonic adeno carcinoma, Burkiff’s lymphoma and viral infection. These show their links with viral disease, neurodevelopment and cancer. It has been shown that they play a key role in melanoma metastasis. These may be

  12. Combinatorics of RNA-RNA interaction

    DEFF Research Database (Denmark)

    Li, Thomas J X; Reidys, Christian

    2012-01-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

  13. STRUCTURILE DE DEFLECTORI, FACTORI DE ÎMBUNĂTĂŢIRE AI HABITATULUI PISCICOL- STUDIU DE CAZ, RÂUL NICOLET (QUEBEC-CANADA

    Directory of Open Access Journals (Sweden)

    Marius DULGHERU

    2010-06-01

    Full Text Available Deflection structures factors for improve the fish habitat. Case study nicolet river (Quebec-Canada. Există câţiva factori care joacă un rol important în definirea calităţii habitatului fizic al râurilor, speciile de peşti manifestând preferinţe pentru anumite elemente hidraulice (viteză, adâncime, tipul sedimentelor etc. Un habitat sănătos este în mod normal caracterizat printr-o succesiune morfologică de vaduri şi adâncuri cu impact în oxigenarea apei, reproducerea şi hrănirea peştilor etc. Datorită importanţei recreaţionale a pescuitului, în Canada există un număr foarte mare de proiecte de îmbunătăţire a habitatului piscicol. In acest sens, deflectorii amplasaţi în albiile cursurilor de apă s-au dovedit a fi metoda cea mai de succes pentru habitatul păstrăvilor. Pe râul Nicolet (Quebec-Canada a fost monitorizată influenţa unor astfel de structuri inginereşti asupra menţinerii în timp a structurii de adânc. Din analiza evoluţiei morfologice şi morfometrice a adâncurilor (prin folosirea diferitelor ridicări topografice succesive din perioada 2000-2007, rezultă faptul că structurile inginereşti au un rol benefic în menţinerea unui habitat propice pentru peşti.

  14. The untranslated regions of classic swine fever virus RNA trigger apoptosis.

    Directory of Open Access Journals (Sweden)

    Wei-Li Hsu

    Full Text Available Classical swine fever virus (CSFV causes a broad range of disease in pigs, from acute symptoms including high fever and hemorrhages, to chronic disease or unapparent infection, depending on the virus strain. CSFV belongs to the genus Pestivirus of the family Flaviviridae. It carries a single-stranded positive-sense RNA genome. An internal ribosomal entry site (IRES in the 5' untranslated region (UTR drives the translation of a single open reading frame encoding a 3898 amino acid long polypeptide chain. The open reading frame is followed by a 3' UTR comprising four highly structured stem-loops. In the present study, a synthetic RNA composed of the 5' and 3' UTRs of the CSFV genome devoid of any viral coding sequence and separated by a luciferase gene cassette (designated 5'UTR-Luc-3'UTR triggered apoptotic cell death as early as 4 h post-transfection. The apoptosis was measured by DNA laddering analysis, TUNEL assay, annexin-V binding determined by flow cytometry, and by analysis of caspase activation. Contrasting with this, only trace DNA laddering was observed in cells transfected with the individual 5' or 3' UTR RNA; even when the 5' UTR and 3' UTR were co-transfected as separate RNA molecules, DNA laddering did not reach the level induced by the chimeric 5'UTR-Luc-3'UTR RNA. Interestingly, RNA composed of the 5'UTR and of stem-loop I of the 3'UTR triggered much stronger apoptosis than the 5' or 3'UTR alone. These results indicate that the 5' and 3' UTRs act together in cis induce apoptosis. We furthered obtained evidence that the UTR-mediated apoptosis required double-stranded RNA and involved translation shutoff possibly through activation of PKR.

  15. Jaume Ferrer de Blanes i Les sentències catòliques del diví poeta Dant. Una mostra de la presència de Dant al renaixement català

    Directory of Open Access Journals (Sweden)

    Josep Llorenç i Blat

    2014-12-01

    Full Text Available Procurador reial i nobiliari, cosmògraf, joier, lapidari, mercader i escriptor, i al capdavall, un ciutadà català honrat, Ferrer (Vidreres, ~1445 – Blanes, 1529 va marxar de ben jove a la cort de Nàpols, al servei del rei Ferran i. Acabada aquesta peripècia italiana va tornar a Blanes al servei del vescomte de Cabrera i de Bas fins que va morir a la mateixa vila al 1529. Un seu criat, disset anys més tard, va editar uns papers esparsos que havia trobat a can Ferrer, les Sentèncias cathòlicas del diví poeta Dant florentí, compilades per lo prudentíssim mossèn Jaume Ferrer de Blanes, que constiueixen un exemple de la presència de Dant al renaixement català. Lord Ferrer (Vidreres,~1445 – Blanes,1529, royal and aristocratic attorney, cosmographer, jewelrer, lapidary, merchant and writer, after all, an honorable Catalan citizen, left early age, at the court of Naples at King Ferdinand’s i service. After this eventful Italian journey he went back to Blanes at the Viscount of Cabrera and Bas’ service, until he died in the same town in 1529. One of his servants, seventeen years later, published some of the documents which were found scattered in Ferrer’s House, Catholic sentences from the divine poet Dante Florentine, compiled by most respectable lord Jaume Ferrer de Blanes, an exemple of the presence of Dante in Catalan Renaissance.

  16. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication.

    Science.gov (United States)

    Niepmann, Michael; Shalamova, Lyudmila A; Gerresheim, Gesche K; Rossbach, Oliver

    2018-01-01

    Hepatitis C virus (HCV) preferentially replicates in the human liver and frequently causes chronic infection, often leading to cirrhosis and liver cancer. HCV is an enveloped virus classified in the genus Hepacivirus in the family Flaviviridae and has a single-stranded RNA genome of positive orientation. The HCV RNA genome is translated and replicated in the cytoplasm. Translation is controlled by the Internal Ribosome Entry Site (IRES) in the 5' untranslated region (5' UTR), while also downstream elements like the cis -replication element (CRE) in the coding region and the 3' UTR are involved in translation regulation. The cis -elements controlling replication of the viral RNA genome are located mainly in the 5'- and 3'-UTRs at the genome ends but also in the protein coding region, and in part these signals overlap with the signals controlling RNA translation. Many long-range RNA-RNA interactions (LRIs) are predicted between different regions of the HCV RNA genome, and several such LRIs are actually involved in HCV translation and replication regulation. A number of RNA cis -elements recruit cellular RNA-binding proteins that are involved in the regulation of HCV translation and replication. In addition, the liver-specific microRNA-122 (miR-122) binds to two target sites at the 5' end of the viral RNA genome as well as to at least three additional target sites in the coding region and the 3' UTR. It is involved in the regulation of HCV RNA stability, translation and replication, thereby largely contributing to the hepatotropism of HCV. However, we are still far from completely understanding all interactions that regulate HCV RNA genome translation, stability, replication and encapsidation. In particular, many conclusions on the function of cis -elements in HCV replication have been obtained using full-length HCV genomes or near-full-length replicon systems. These include both genome ends, making it difficult to decide if a cis -element in question acts on HCV

  17. RNA modifications by oxidation

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

    2012-01-01

    to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

  18. Working with RNA

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

  19. Acute Endoplasmic Reticulum Stress-Independent Unconventional Splicing of XBP1 mRNA in the Nucleus of Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Yuanyuan Wang

    2015-06-01

    Full Text Available The regulation of expression of X-box-binding protein-1 (XBP1, a transcriptional factor, involves an unconventional mRNA splicing that removes the 26 nucleotides intron. In contrast to the conventional splicing that exclusively takes place in the nucleus, determining the location of unconventional splicing still remains controversial. This study was designed to examine whether the unconventional spicing of XBP1 mRNA could occur in the nucleus and its possible biological relevance. We use RT-PCR reverse transcription system and the expand high fidelity PCR system to detect spliced XBP1 mRNA, and fraction cells to determine the location of the unconventional splicing of XBP1 mRNA. We employ reporter constructs to show the presence of unconventional splicing machinery in mammal cells independently of acute endoplasmic reticulum (ER stress. Our results reveal the presence of basal unconventional splicing of XBP1 mRNA in the nucleus that also requires inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α and can occur independently of acute ER stress. Furthermore, we confirm that acute ER stress induces the splicing of XBP1 mRNA predominantly occurring in the cytoplasm, but it also promotes the splicing in the nucleus. The deletion of 5′-nucleotides in XBP1 mRNA significantly increases its basal unconventional splicing, suggesting that the secondary structure of XBP1 mRNA may determine the location of unconventional splicing. These results suggest that the unconventional splicing of XBP1 mRNA can take place in the nucleus and/or cytoplasm, which possibly depends on the elaborate regulation. The acute ER stress-independent unconventional splicing in the nucleus is most likely required for the maintaining of day-to-day folding protein homeostasis.

  20. Accidental release of iodine-131 by IRE at Fleurus: back experience of Belgium safety authority; Rejet accidentel d'iode-131 par l'IRE sur le site de Fleurus: retour d'experience de l'autorite de surete belge

    Energy Technology Data Exchange (ETDEWEB)

    Vandecasteele, C.M.; Sonck, M. [AFCN - Agence federale de controle nucleaire, Bruxelles (Belgium); Degueldre, D. [Bel V, Anderlecht (Belgium)

    2011-04-15

    The IRE (National institute for radioelements) produces radionuclides for nuclear medicine from highly enriched uranium irradiated targets. On 22/08/2008, fresh production wastes were transferred into an almost empty decay tank. The mixing of these liquids led to the release of approximately 47 GBq of molecular iodine-131 into the atmosphere. The first conservative assessments of the radiological consequences did not require taking direct protective actions for the population, such as sheltering or stable iodine intake. However, the estimated iodine-131 deposits could locally reach or exceed the derived reference levels for the contamination of milk (4 kBq/m2) and leafy vegetables (10 kBq/m2). For this reason, and because there was a threat of a further release, the federal emergency plan was activated on 28/08 and the population potentially concerned was recommended to avoid consumption of locally produced fruits, vegetables and fresh milk. These protective actions were lifted on 7/09 and the emergency plan was lifted on 12/09. The main lesson learned from this event concerns the paramount importance of the rapid exchange of information that is as accurate and complete as possible between the different stakeholders: from the operator up to the population, through federal and local authorities. (authors)

    1. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication

      Directory of Open Access Journals (Sweden)

      Michael Niepmann

      2018-03-01

      Full Text Available Hepatitis C virus (HCV preferentially replicates in the human liver and frequently causes chronic infection, often leading to cirrhosis and liver cancer. HCV is an enveloped virus classified in the genus Hepacivirus in the family Flaviviridae and has a single-stranded RNA genome of positive orientation. The HCV RNA genome is translated and replicated in the cytoplasm. Translation is controlled by the Internal Ribosome Entry Site (IRES in the 5′ untranslated region (5′ UTR, while also downstream elements like the cis-replication element (CRE in the coding region and the 3′ UTR are involved in translation regulation. The cis-elements controlling replication of the viral RNA genome are located mainly in the 5′- and 3′-UTRs at the genome ends but also in the protein coding region, and in part these signals overlap with the signals controlling RNA translation. Many long-range RNA–RNA interactions (LRIs are predicted between different regions of the HCV RNA genome, and several such LRIs are actually involved in HCV translation and replication regulation. A number of RNA cis-elements recruit cellular RNA-binding proteins that are involved in the regulation of HCV translation and replication. In addition, the liver-specific microRNA-122 (miR-122 binds to two target sites at the 5′ end of the viral RNA genome as well as to at least three additional target sites in the coding region and the 3′ UTR. It is involved in the regulation of HCV RNA stability, translation and replication, thereby largely contributing to the hepatotropism of HCV. However, we are still far from completely understanding all interactions that regulate HCV RNA genome translation, stability, replication and encapsidation. In particular, many conclusions on the function of cis-elements in HCV replication have been obtained using full-length HCV genomes or near-full-length replicon systems. These include both genome ends, making it difficult to decide if a cis-element in

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

      Directory of Open Access Journals (Sweden)

      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.

    3. Methods for RNA Analysis

      DEFF Research Database (Denmark)

      Olivarius, Signe

      of the transcriptome, 5’ end capture of RNA is combined with next-generation sequencing for high-throughput quantitative assessment of transcription start sites by two different methods. The methods presented here allow for functional investigation of coding as well as noncoding RNA and contribute to future...... RNAs rely on interactions with proteins, the establishment of protein-binding profiles is essential for the characterization of RNAs. Aiming to facilitate RNA analysis, this thesis introduces proteomics- as well as transcriptomics-based methods for the functional characterization of RNA. First, RNA...

    4. Cytoplasmic Z-RNA

      International Nuclear Information System (INIS)

      Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

      1987-01-01

      Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

    5. La importancia de los proyectos y redes innovadoras para el avance de la Enseñanza de las Ciencias: El caso de un profesor de la Red IRES

      Directory of Open Access Journals (Sweden)

      Gabriela Delord

      2017-01-01

      Full Text Available Este articulo presenta la descripción y análisis del Modelo de Investigación en la Escuela que fundamenta el Proyecto IRES (Investigación y Renovación Escolar y el análisis en profundidad de una entrevista a un maestro en el área de ciencias que pertenece a la red de este proyecto: Red IRES. Los resultados ponen en evidencia la importancia que ha tenido para este profesor la pertenencia al proyecto y al colectivo de docentes que lo impulsan, así como la pertinencia del modelo para sustentar los cambios introducidos en el aula. Destaca también la relevancia, para su evolución profesional, del diálogo entre docentes de todos los niveles del sistema educativo, incluida la universidad. Proponemos que estas experiencias sean más reconocidas y valoradas en el ámbito de la Enseñanza de las Ciencias y en las instancias gubernamentales.

    6. 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.

    7. Tratamento cirúrgico da comunicação interventricular e rotura da parede livre do ventrículo esquerdo após infarto agudo do miocárdio e da disjunção atrioventricular após troca da valva mitral, empregando o dispositivo de isolamento ventricular (DIV:: estudo experimental

      Directory of Open Access Journals (Sweden)

      BERNARDES Rodrigo de Castro

      1997-01-01

      Full Text Available A rotura de parede livre do ventrículo esquerdo (VE e a comunicação interventricular (CIV por rotura de septo são dois eventos catastróficos que podem complicar a evolução do infarto agudo do miocárdio (IAM. A oportunidade do tratamento cirúrgico, o acesso à CIV e a técnica cirúrgica ideal para o tratamento desta complicação são muito discutidos na literatura (1-5. Os resultados, porém, são quase unânimes: alta taxa de mortalidade. A rotura da parede posterior do ventrículo esquerdo pós troca da valva mitral (disjunção atrioventricular não é uma complicação rara, sendo quase sempre fatal (6, 7. O objetivo de nosso trabalho é discutir a alternativa de tratamento cirúrgico destas graves lesões, sem o manuseio direto do músculo cardíaco friável, necrozado. Desenvolvemos e testamos em animais (carneiros o dispositivo de isolamento ventricular (DIV. No período de 12/7/95 a 10/1/96, operamos 12 carneiros adultos para o implante do DIV. No primeiro período de aprendizado da técnica de implante e de desenvolvimento da prótese, operamos 7 animais, sem sucesso. No segundo período operamos 5 animais, já com o DIV completamente desenvolvido e com a técnica cirúrgica já padronizada; obtivemos sucesso com todos os animais, sobrevivendo com bom débito cardíaco, constatado ao ecocardiograma e à ventriculografia. Após 20 dias de sobrevida, os animais foram submetidos a ventriculografia e ecocardiografia. Observamos o completo isolamento da cavidade ventricular com o emprego do DIV, obtendo o tratamento provisório destas graves lesões.

    8. RNA decay by messenger RNA interferases

      DEFF Research Database (Denmark)

      Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

      2008-01-01

      Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mR...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.......Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...

    9. Topology of RNA-RNA interaction structures

      DEFF Research Database (Denmark)

      Andersen, Jørgen Ellegaard; Huang, Fenix Wenda; Penner, Robert

      2012-01-01

      Abstract The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist...

    10. RNA Localization in Astrocytes

      DEFF Research Database (Denmark)

      Thomsen, Rune

      2012-01-01

      , regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA......Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

    11. Measurement of imino {sup 1}H-{sup 1}H residual dipolar couplings in RNA

      Energy Technology Data Exchange (ETDEWEB)

      Latham, Michael P. [University of Toronto, Department of Molecular Genetics (Canada); Pardi, Arthur [University of Colorado, Department of Chemistry and Biochemistry, 215 UCB (United States)], E-mail: arthur.pardi@colorado.edu

      2009-02-15

      Imino {sup 1}H-{sup 15}N residual dipolar couplings (RDCs) provide additional structural information that complements standard {sup 1}H-{sup 1}H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino {sup 1}H-{sup 1}H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA{sup Val} using a BEST-Jcomp-HMQC2 experiment. {sup 1}H-{sup 1}H RDCs are observed between the imino protons in G-U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino {sup 1}H-{sup 1}H RDCs complement standard {sup 1}H-{sup 15}N RDCs because the {sup 1}H-{sup 1}H vectors generally point along the helical axis, roughly perpendicular to {sup 1}H-{sup 15}N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by {approx}3.5-fold over the standard experiment. The ability to measure imino {sup 1}H-{sup 1}H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs.

    12. Tat-haFGF14–154 Upregulates ADAM10 to Attenuate the Alzheimer Phenotype of APP/PS1 Mice through the PI3K-CREB-IRE1α/XBP1 Pathway

      Directory of Open Access Journals (Sweden)

      Tian Meng

      2017-06-01

      Full Text Available Acid fibroblast growth factor (aFGF has shown neuroprotection in Alzheimer’s disease (AD models in previous studies, yet its mechanism is still uncertain. Here we report that the efficacy of Tat-haFGF14–154 is markedly increased when loaded cationic liposomes for intranasal delivery are intranasally administered to APP/PS1 mice. Our results demonstrated that liposomal Tat-haFGF14–154 treatment significantly ameliorated behavioral deficits, relieved brain Aβ burden, and increased the expression and activity of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10 in the brain. Tat-haFGF14–154 antagonized Aβ1–42-induced cell death and structural damage in rat primary neurons in an ADAM10-dependent manner, which, in turn, was promoted by the activation of XBP1 splicing and modulated by the PI3K-CREB pathway. Both knockdown of ADAM10 and inhibition of PI3K (LY294002 negated Tat-haFGF14–154 rescue. Thus, Tat-haFGF14–154 activates the IRE1α/XBP1 pathway of the unfolded protein response (UPR against the endoplasmic reticulum (ER stress induced by Aβ, and, subsequently, the nuclear translocation of spliced XBP1 (XBP1s promotes transcription of ADAM10. These results highlight the important role of ADAM10 and its activation through the PI3K-CREB-IRE1α/XBP1 pathway as a key factor in the mechanism of neuroprotection for Tat-haFGF14–154.

    13. Assembling RNA Nanoparticles.

      Science.gov (United States)

      Xiao, Shou-Jun

      2017-01-01

      RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

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

      Directory of Open Access Journals (Sweden)

      Sung Un eHuh

      2013-12-01

      Full Text Available 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 binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

    15. Shapes of interacting RNA complexes

      DEFF Research Database (Denmark)

      Fu, Benjamin Mingming; Reidys, Christian

      2014-01-01

      Shapes of interacting RNA complexes are studied using a filtration via their topological genus. A shape of an RNA complex is obtained by (iteratively) collapsing stacks and eliminating hairpin loops.This shape-projection preserves the topological core of the RNA complex and for fixed topological...... genus there are only finitely many such shapes. Our main result is a new bijection that relates the shapes of RNA complexes with shapes of RNA structures. This allows to compute the shape polynomial of RNA complexes via the shape polynomial of RNA structures. We furthermore present a linear time uniform...... sampling algorithm for shapes of RNA complexes of fixed topological genus....

    16. Remote Network Access (RNA)

      National Research Council Canada - National Science Library

      2002-01-01

      .... Remote Network Access (RNA) includes or is associated with all communication devices/software, firewalls, intrusion detection systems and virus protection applications to ensure security of the OIG, DoD, Network from remote...

    17. RNA/PNA Approach

      Indian Academy of Sciences (India)

      In this approach we want to develop structural analogue of the leader that might have higher affinity towards the Phosphoprotein, but would impair the dimerization process and viral leader RNA binding.

    18. Switching off small RNA regulation with trap-mRNA

      DEFF Research Database (Denmark)

      Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

      2009-01-01

      to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

    19. Papel dos avós: apoio oferecido aos netos antes e após situações de separação/divórcio dos pais The grandparents role: support offered to the grandchildren before and after situations of parent's separation/divorce

      Directory of Open Access Journals (Sweden)

      Mayeve Rochane Gerônimo Leite Araújo

      2002-01-01

      Full Text Available A presente pesquisa teve como objetivo averiguar os tipos de apoio oferecidos pelos avós aos netos antes e após situações de separação/divórcio dos pais, bem como a existência ou não de alterações nas formas de apoio. Foram considerados dois tipos de apoio: o emocional e o instrumental, sob a influência das seguintes variáveis: distância geográfica, custódia parental, vinculação materna ou paterna, idade, situação empregatícia e estado civil dos avós. Participaram da mesma 30 avós que não moravam com os netos, antes nem após a separação/divórcio dos pais. O instrumento utilizado foi um questionário contendo itens que atendiam aos objetivos da pesquisa, bem como os dados sócio-demográficos. Os resultados indicaram uma preferência, por parte dos avós, pelas atividades do tipo emocional, notando-se um aumento, em geral, após a separação/divórcio, nas seguintes atividades: dar conselhos, transmitir informações sobre a família, telefonar e uma diminuição na atividade visitar.The present research aimed to discover the types of support offered by the grandparents to grandchildren, before and after the parents' separation/divorce, as well as the possible changes in the forms of support. Two types of support were analyzed: the emotional and the instrumental, considering the influence of the following variables: geographical distance, custody, maternal or paternal link, age, marital status and grandparents' job situation. The subjects were 30 grandparents who didn't live with their grandchildren, before or after the parent's separation/divorce, were selected. The instrument used was a questionnaire with items which met the objectives of the research. The social demographic factors were also considered. Results indicated a preference for activities of the emotional type, with a general increase, after the separation/divorce, in activities such as: giving advice, transmitting information concerning the family, and

    20. A ribosome without RNA

      Directory of Open Access Journals (Sweden)

      Harold S Bernhardt

      2015-11-01

      Full Text Available It was Francis Crick who first asked why the ribosome contains so much RNA, and discussed the implications of this for the direct flow of genetic information from DNA to protein. Remarkable advances in our understanding of the ribosome and protein synthesis, including the recent publication of two mammalian mitochondrial ribosome structures, have shed new light on this intriguing aspect of evolution in molecular biology. We examine here whether RNA is indispensable for coded protein synthesis, or whether an all-protein ‘ribosome’ (or ‘synthosome’ might be possible, with a protein enzyme catalyzing peptide synthesis, and release factor-like protein adaptors able to read a message composed of deoxyribonucleotides. We also compare the RNA world hypothesis with the alternative ‘proteins first’ hypothesis in terms of their different understandings of the evolution of the ribosome, and whether this might have been preceded by an ancestral form of nonribosomal peptide synthesis catalyzed by protein enzymes.

    1. Pyrite footprinting of RNA

      International Nuclear Information System (INIS)

      Schlatterer, Jörg C.; Wieder, Matthew S.; Jones, Christopher D.; Pollack, Lois; Brenowitz, Michael

      2012-01-01

      Highlights: ► RNA structure is mapped by pyrite mediated · OH footprinting. ► Repetitive experiments can be done in a powdered pyrite filled cartridge. ► High · OH reactivity of nucleotides imply dynamic role in Diels–Alderase catalysis. -- Abstract: In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ( · OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS 2 ) can produce sufficient · OH to footprint DNA. The 49-nt Diels–Alder RNA enzyme catalyzes the C–C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme’s active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels–Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to · OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop’s flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated · OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

    2. RNA Regulation of Estrogen

      Science.gov (United States)

      2010-08-01

      Berglund, Rodger Voelker, Paul Barber and Julien Diegel 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING...estrogen  receptors  [reviewed  in  (3,  4)],  also   functions   by  interacting  directly  with  RNA  to  alter  RNA...Mog myelin oligodendrocyte glycoprotein 6.06 207115_x_at mbtd1 mbt domain containing 1 6.06 208004_at Prol1 proline rich, lacrimal 1 6.06 205247_at

    3. RNA Regulation by Estrogen

      Science.gov (United States)

      2011-08-01

      Julien Diegel, Amy Mahady, and Micah Bodner 5e. TASK NUMBER E-Mail: aberglund@molbio.uoregon.edu 5f. WORK UNIT NUMBER 7. PERFORMING...4)],  also   functions   by  interacting  directly  with  RNA  to  alter  RNA  processing  events  such  as  splicing...1 6.06 208004_at Prol1 proline rich, lacrimal 1 6.06 205247_at NOTCH4 Notch homolog 4 (Drosophila) 6.06 211203_s_at Cntn1 contactin 1 6.06 220689_at

    4. Sensing of RNA viruses

      DEFF Research Database (Denmark)

      Jensen, Søren; Thomsen, Allan Randrup

      2012-01-01

      pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways.......Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...

    5. RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

      Directory of Open Access Journals (Sweden)

      Andronescu Mirela

      2008-08-01

      Full Text Available Abstract Background The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures. Results In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND. Conclusion RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at http://www.rnasoft.ca/strand.

    6. Studying RNA-protein interactions in vivo by RNA immunoprecipitation

      DEFF Research Database (Denmark)

      Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

      2011-01-01

      and have significant effects on gene expression. RNA immunoprecipitation (RIP) is a powerful technique used to detect direct and indirect interactions between individual proteins and specific RNA molecules in vivo. Here, we describe RIP methods for both yeast and mammalian cells.......The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases...

    7. Branched RNA: A New Architecture for RNA Interference

      Directory of Open Access Journals (Sweden)

      Anna Aviñó

      2011-01-01

      Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

    8. The RNA gene information: retroelement-microRNA entangling as the RNA quantum code.

      Science.gov (United States)

      Fujii, Yoichi Robertus

      2013-01-01

      MicroRNA (miRNA) and retroelements may be a master of regulator in our life, which are evolutionally involved in the origin of species. To support the Darwinism from the aspect of molecular evolution process, it has tremendously been interested in the molecular information of naive RNA. The RNA wave model 2000 consists of four concepts that have altered from original idea of the miRNA genes for crosstalk among embryonic stem cells, their niche cells, and retroelements as a carrier vesicle of the RNA genes. (1) the miRNA gene as a mobile genetic element induces transcriptional and posttranscriptional silencing via networking-processes (no hierarchical architecture); (2) the RNA information supplied by the miRNA genes expands to intracellular, intercellular, intraorgan, interorgan, intraspecies, and interspecies under the cycle of life into the global environment; (3) the mobile miRNAs can self-proliferate; and (4) cells contain two types information as resident and genomic miRNAs. Based on RNA wave, we have developed an interest in investigation of the transformation from RNA information to quantum bits as physicochemical characters of RNA with the measurement of RNA electron spin. When it would have been given that the fundamental bases for the acquired characters in genetics can be controlled by RNA gene information, it may be available to apply for challenging against RNA gene diseases, such as stress-induced diseases.

    9. Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl2.

      Science.gov (United States)

      Damiano, Fabrizio; Testini, Mariangela; Tocci, Romina; Gnoni, Gabriele V; Siculella, Luisa

      2018-04-01

      Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl 2 , up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

    10. Plant RNA Regulatory Network and RNA Granules in Virus Infection

      Directory of Open Access Journals (Sweden)

      Kristiina Mäkinen

      2017-12-01

      Full Text Available Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and

    11. Plant RNA Regulatory Network and RNA Granules in Virus Infection.

      Science.gov (United States)

      Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

      2017-01-01

      Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual

    12. RNA-Catalyzed Polymerization and Replication of RNA

      Science.gov (United States)

      Horning, D. P.; Samantha, B.; Tjhung, K. F.; Joyce, G. F.

      2017-07-01

      In an effort to reconstruct RNA-based life, in vitro evolution was used to obtain an RNA polymerase ribozyme that can synthesize a variety of complex functional RNAs and can catalyze the exponential amplification of short RNAs.

    13. Natural RNA circles function as efficient microRNA sponges

      DEFF Research Database (Denmark)

      Hansen, Thomas Birkballe; Jensen, Trine I; Clausen, Bettina Hjelm

      2013-01-01

      MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called comp......MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so......-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more...... sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA....

    14. Strategies underlying RNA silencing suppression by negative strand RNA viruses

      NARCIS (Netherlands)

      Hemmes, J.C.

      2007-01-01

      The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double

    15. RNA Interference - Towards RNA becoming a Medicine -42 ...

      Indian Academy of Sciences (India)

      research. A brief history of the development ofRNAi is shown in. Box 2. Mechanism of ... new RNA strand using target RNA as the template and thereby converting it ... thought to excise precursor stRNA from their -70 nt stem loop precursor to ...

    16. Semiautomated improvement of RNA alignments

      DEFF Research Database (Denmark)

      Andersen, Ebbe Sloth; Lind-Thomsen, Allan; Knudsen, Bjarne

      2007-01-01

      connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database...... and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster......: the mir-399 RNA, vertebrate telomase RNA (vert-TR), bacterial transfer-messenger RNA (tmRNA), and the signal recognition particle (SRP) RNA. The general use of the method is illustrated by the ability to accommodate pseudoknots and handle even large and divergent RNA families. The open architecture...

    17. Comparative RNA genomics

      DEFF Research Database (Denmark)

      Backofen, Rolf; Gorodkin, Jan; Hofacker, Ivo L.

      2018-01-01

      Over the last two decades it has become clear that RNA is much more than just a boring intermediate in protein expression. Ancient RNAs still appear in the core information metabolism and comprise a surprisingly large component in bacterial gene regulation. A common theme with these types of mostly...... small RNAs is their reliance of conserved secondary structures. Large scale sequencing projects, on the other hand, have profoundly changed our understanding of eukaryotic genomes. Pervasively transcribed, they give rise to a plethora of large and evolutionarily extremely flexible noncoding RNAs...... that exert a vastly diverse array of molecule functions. In this chapter we provide a—necessarily incomplete—overview of the current state of comparative analysis of noncoding RNAs, emphasizing computational approaches as a means to gain a global picture of the modern RNA world....

    18. MicroRNA from tuberculosis RNA: A bioinformatics study

      OpenAIRE

      Wiwanitkit, Somsri; Wiwanitkit, Viroj

      2012-01-01

      The role of microRNA in the pathogenesis of pulmonary tuberculosis is the interesting topic in chest medicine at present. Recently, it was proposed that the microRNA can be a useful biomarker for monitoring of pulmonary tuberculosis and might be the important part in pathogenesis of disease. Here, the authors perform a bioinformatics study to assess the microRNA within known tuberculosis RNA. The microRNA part can be detected and this can be important key information in further study of the p...

    19. RNA binding and replication by the poliovirus RNA polymerase

      International Nuclear Information System (INIS)

      Oberste, M.S.

      1988-01-01

      RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

    20. Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

      International Nuclear Information System (INIS)

      Bodkin, D.K.

      1985-01-01

      RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

    1. RNA-SSPT: RNA Secondary Structure Prediction Tools.

      Science.gov (United States)

      Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

      2013-01-01

      The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes.

    2. Regulation of MicroRNA-378 expression in mature human adipose ...

      African Journals Online (AJOL)

      ... To investigate the effects of adiponectin (ADPN), free fatty acids (FFAs), growth hormone ... ADPN, and DEX, the interaction of which may be involved in the pathogenesis of obesity-induced ..... that two key ER stress sensors, PERK and IRE-.

    3. RNA meets disease in paradise.

      Science.gov (United States)

      Winter, Julia; Roth, Anna; Diederichs, Sven

      2011-01-01

      Getting off the train in Jena-Paradies, 60 participants joined for the 12 (th) Young Scientist Meeting of the German Society for Cell Biology (DGZ) entitled "RNA & Disease". Excellent speakers from around the world, graduate students, postdocs and young group leaders enjoyed a meeting in a familiar atmosphere to exchange inspiring new data and vibrant scientific discussions about the fascinating history and exciting future of non-coding RNA research including microRNA, piRNA and long non-coding RNA as well as their function in cancer, diabetes and neurodegenerative diseases.

    4. From "Cellular" RNA to "Smart" RNA: Multiple Roles of RNA in Genome Stability and Beyond.

      Science.gov (United States)

      Michelini, Flavia; Jalihal, Ameya P; Francia, Sofia; Meers, Chance; Neeb, Zachary T; Rossiello, Francesca; Gioia, Ubaldo; Aguado, Julio; Jones-Weinert, Corey; Luke, Brian; Biamonti, Giuseppe; Nowacki, Mariusz; Storici, Francesca; Carninci, Piero; Walter, Nils G; Fagagna, Fabrizio d'Adda di

      2018-03-30

      Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

    5. dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

      Directory of Open Access Journals (Sweden)

      Eliane F. Meurs

      2012-10-01

      Full Text Available The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2a. As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2a kinase, its participation in the regulation of the NF-kB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV. PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2a-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

    6. Transfer RNA and human disease

      Directory of Open Access Journals (Sweden)

      Jamie A Abbott

      2014-06-01

      Full Text Available Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA genes are hotspots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase, mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers, and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing. Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

    7. Transfer RNA and human disease.

      Science.gov (United States)

      Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

      2014-01-01

      Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

    8. RNA Thermodynamic Structural Entropy.

      Science.gov (United States)

      Garcia-Martin, Juan Antonio; Clote, Peter

      2015-01-01

      Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

    9. RNA Thermodynamic Structural Entropy.

      Directory of Open Access Journals (Sweden)

      Juan Antonio Garcia-Martin

      Full Text Available Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs. However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

    10. Identifying microRNA/mRNA dysregulations in ovarian cancer.

      Science.gov (United States)

      Miles, Gregory D; Seiler, Michael; Rodriguez, Lorna; Rajagopal, Gunaretnam; Bhanot, Gyan

      2012-03-27

      MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms. Our findings identify

    11. RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

      International Nuclear Information System (INIS)

      Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E.; Eghbalnia, Hamid R.

      2012-01-01

      The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ( 1 H– 15 N 2D HMQC) and proton–proton nuclear Overhauser enhancement spectroscopy ( 1 H– 1 H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino resonances for a

    12. RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

      Energy Technology Data Exchange (ETDEWEB)

      Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E. [National Magnetic Resonance Facility at Madison (United States); Eghbalnia, Hamid R., E-mail: eghbalhd@uc.edu [University of Cincinnati, Department of Molecular and Cellular Physiology (United States)

      2012-04-15

      The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ({sup 1}H-{sup 15}N 2D HMQC) and proton-proton nuclear Overhauser enhancement spectroscopy ({sup 1}H-{sup 1}H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino

    13. RNA SURVEILLANCE– AN EMERGING ROLE FOR RNA REGULATORY NETWORKS IN AGING

      OpenAIRE

      Montano, Monty; Long, Kimberly

      2010-01-01

      In this review, we describe recent advances in the field of RNA regulatory biology and relate these advances to aging science. We introduce a new term, RNA surveillance, an RNA regulatory process that is conserved in metazoans, and describe how RNA surveillance represents molecular cross-talk between two emerging RNA regulatory systems – RNA interference and RNA editing. We discuss how RNA surveillance mechanisms influence mRNA and microRNA expression and activity during lifespan. Additionall...

    14. On RNA-RNA interaction structures of fixed topological genus.

      Science.gov (United States)

      Fu, Benjamin M M; Han, Hillary S W; Reidys, Christian M

      2015-04-01

      Interacting RNA complexes are studied via bicellular maps using a filtration via their topological genus. Our main result is a new bijection for RNA-RNA interaction structures and a linear time uniform sampling algorithm for RNA complexes of fixed topological genus. The bijection allows to either reduce the topological genus of a bicellular map directly, or to lose connectivity by decomposing the complex into a pair of single stranded RNA structures. Our main result is proved bijectively. It provides an explicit algorithm of how to rewire the corresponding complexes and an unambiguous decomposition grammar. Using the concept of genus induction, we construct bicellular maps of fixed topological genus g uniformly in linear time. We present various statistics on these topological RNA complexes and compare our findings with biological complexes. Furthermore we show how to construct loop-energy based complexes using our decomposition grammar. Copyright © 2015 Elsevier Inc. All rights reserved.

    15. antaRNA: ant colony-based RNA sequence design.

      Science.gov (United States)

      Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

      2015-10-01

      RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found ,: inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology ,: reliable RNA sequence design becomes a crucial step to generate novel biochemical components. In this article ,: the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution ,: specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. http://www.bioinf.uni-freiburg.de/Software/antaRNA CONTACT: backofen@informatik.uni-freiburg.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

    16. RNA İNTERFERANS (RNAİ)

      OpenAIRE

      GÜNDOĞDU, Ramazan; ÇELİK, Venhar

      2009-01-01

      RNA interferans, uygun çift zincirli RNA’nın hücreye girdiği zaman, endojenik komplementer mRNA dizisinin parçalanmasına yol açan, transkripsiyon sonrası gen susturma mekanizmasıdır. RNA interferans, Dicer adı verilen bir RNase III enzimi tarafından çift zincirli RNA’nın küçük engelleyici RNA’lara (siRNA) kesilmesi ile başlamaktadır. Bu siRNA’lar daha sonra, bir multiprotein-RNA nükleaz kompleksi olan, RNA- indükleyici baskılama kompleksine (RISC) bağlanır. RISC, siRNA’ları komplementer mRNA’...

    17. Radiation sensitivity of messenger RNA

      International Nuclear Information System (INIS)

      Ponta, H.; Pfennig-Yeh, M.L.; Herrlich, P.; Karlsruhe Univ.; Wagner, E.F.; Schweiger, M.

      1979-01-01

      Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research. (orig.) [de

    18. Radiation sensitivity of messenger RNA

      Energy Technology Data Exchange (ETDEWEB)

      Ponta, H; Pfennig-Yeh, M L; Herrlich, P [Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und Toxikologie von Spaltstoffen; Karlsruhe Univ. (TH) (Germany, F.R.). Inst. fuer Genetik); Wagner, E F; Schweiger, M [Innsbruck Univ. (Austria). Inst. fuer Biochemie

      1979-08-01

      Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research.

    19. RNase-assisted RNA chromatography

      Science.gov (United States)

      Michlewski, Gracjan; Cáceres, Javier F.

      2010-01-01

      RNA chromatography combined with mass spectrometry represents a widely used experimental approach to identify RNA-binding proteins that recognize specific RNA targets. An important drawback of most of these protocols is the high background due to direct or indirect nonspecific binding of cellular proteins to the beads. In many cases this can hamper the detection of individual proteins due to their low levels and/or comigration with contaminating proteins. Increasing the salt concentration during washing steps can reduce background, but at the cost of using less physiological salt concentrations and the likely loss of important RNA-binding proteins that are less stringently bound to a given RNA, as well as the disassembly of protein or ribonucleoprotein complexes. Here, we describe an improved RNA chromatography method that relies on the use of a cocktail of RNases in the elution step. This results in the release of proteins specifically associated with the RNA ligand and almost complete elimination of background noise, allowing a more sensitive and thorough detection of RNA-binding proteins recognizing a specific RNA transcript. PMID:20571124

    20. RNA interference in Lepidoptera

      DEFF Research Database (Denmark)

      Terenius, Ole; Papanicolaou, Alexie; Garbutt, Jennie S.

      2011-01-01

      in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our...... experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi...... is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success...

    1. A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

      Directory of Open Access Journals (Sweden)

      Lucile Warter

      Full Text Available GB virus B (GBV-B is closely related to hepatitis C virus (HCV, infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs of these viruses fold into four similar structured domains (I-IV, with domains II-III-IV comprising the viral internal ribosomal entry site (IRES. We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC genome (within the 3'NTR, upstream of the poly(U tract, and NS5A coding sequence are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.

    2. Concepts and introduction to RNA bioinformatics

      DEFF Research Database (Denmark)

      Gorodkin, Jan; Hofacker, Ivo L.; Ruzzo, Walter L.

      2014-01-01

      RNA bioinformatics and computational RNA biology have emerged from implementing methods for predicting the secondary structure of single sequences. The field has evolved to exploit multiple sequences to take evolutionary information into account, such as compensating (and structure preserving) base...... for interactions between RNA and proteins.Here, we introduce the basic concepts of predicting RNA secondary structure relevant to the further analyses of RNA sequences. We also provide pointers to methods addressing various aspects of RNA bioinformatics and computational RNA biology....

    3. Identification of Subtype Specific miRNA-mRNA Functional Regulatory Modules in Matched miRNA-mRNA Expression Data: Multiple Myeloma as a Case

      OpenAIRE

      Zhang, Yunpeng; Liu, Wei; Xu, Yanjun; Li, Chunquan; Wang, Yingying; Yang, Haixiu; Zhang, Chunlong; Su, Fei; Li, Yixue; Li, Xia

      2015-01-01

      Identification of miRNA-mRNA modules is an important step to elucidate their combinatorial effect on the pathogenesis and mechanisms underlying complex diseases. Current identification methods primarily are based upon miRNA-target information and matched miRNA and mRNA expression profiles. However, for heterogeneous diseases, the miRNA-mRNA regulatory mechanisms may differ between subtypes, leading to differences in clinical behavior. In order to explore the pathogenesis of each subtype, it i...

    4. Bifurcations in the interplay of messenger RNA, protein and nonprotein coding RNA

      International Nuclear Information System (INIS)

      Zhdanov, Vladimir P

      2008-01-01

      The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-protein association resulting in suppression of the protein regulatory activity or (ii) ncRNA-mRNA association resulting in degradation of the miRNA-mRNA complex. The kinetic models describing these two scenarios are found to predict bistability provided that protein suppresses the ncRNA formation

    5. 34A, miRNA-944, miRNA-101 and miRNA-218 in cervical cancer

      African Journals Online (AJOL)

      RNAs (21 - 24 nucleotides in length) that are critical for many important processes such as development, ... RNA extraction and reverse transcription. Total RNA was extracted from each of the experimental groups using ... used as an endogenous control to normalize the expression of miRNA-143, miRNA-34A, miRNA-.

    6. Nuclear Export of Messenger RNA

      Directory of Open Access Journals (Sweden)

      Jun Katahira

      2015-03-01

      Full Text Available Transport of messenger RNA (mRNA from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex.

    7. RNA viruses in the sea.

      Science.gov (United States)

      Lang, Andrew S; Rise, Matthew L; Culley, Alexander I; Steward, Grieg F

      2009-03-01

      Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.

    8. Nuclear Export of Messenger RNA

      Science.gov (United States)

      Katahira, Jun

      2015-01-01

      Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

    9. Transfecting Human Monocytes with RNA.

      Science.gov (United States)

      Dannull, Jens; Nair, Smita K

      2016-01-01

      Targeting monocytes as a delivery system for drugs or nucleic acids, and thereby harnessing their natural tissue-infiltrating capacity, has become an area of intense investigation in both basic and clinical research. Herein we describe an efficient method to deliver mRNA (messenger RNA) or siRNA (small interfering RNA) into human monocytes by electroporation. This method can be applied in the laboratory to monocytes isolated via magnetic bead-based techniques, or in a clinical setting using monocytes that were collected via counterflow centrifugation elutriation using the Elutra(®) Cell Separation System. We further demonstrate that electroporation of monocytes with RNA represents a robust and highly relevant approach to modify monocytes for cell-based therapies. Last, the procedure described can readily be adapted to monocytes from different species, hence facilitating research in animal models.

    10. Fast prediction of RNA-RNA interaction using heuristic algorithm.

      Science.gov (United States)

      Montaseri, Soheila

      2015-01-01

      Interaction between two RNA molecules plays a crucial role in many medical and biological processes such as gene expression regulation. In this process, an RNA molecule prohibits the translation of another RNA molecule by establishing stable interactions with it. Some algorithms have been formed to predict the structure of the RNA-RNA interaction. High computational time is a common challenge in most of the presented algorithms. In this context, a heuristic method is introduced to accurately predict the interaction between two RNAs based on minimum free energy (MFE). This algorithm uses a few dot matrices for finding the secondary structure of each RNA and binding sites between two RNAs. Furthermore, a parallel version of this method is presented. We describe the algorithm's concurrency and parallelism for a multicore chip. The proposed algorithm has been performed on some datasets including CopA-CopT, R1inv-R2inv, Tar-Tar*, DIS-DIS, and IncRNA54-RepZ in Escherichia coli bacteria. The method has high validity and efficiency, and it is run in low computational time in comparison to other approaches.

    11. The RNA synthesis machinery of negative-stranded RNA viruses

      Energy Technology Data Exchange (ETDEWEB)

      Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

      2015-05-15

      The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

    12. The RNA synthesis machinery of negative-stranded RNA viruses

      International Nuclear Information System (INIS)

      Ortín, Juan; Martín-Benito, Jaime

      2015-01-01

      The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes

    13. The 5'-poly(A leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.

      Directory of Open Access Journals (Sweden)

      Pragyesh Dhungel

      2017-08-01

      Full Text Available The poly(A leader at the 5'-untranslated region (5'-UTR is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs. These poly(A leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5'-poly(A leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5'-poly(A leader with 12 residues was optimal. Because the most frequent lengths of the 5'-poly(A leaders are 8-12 residues, the result suggests that the poly(A leader has been evolutionarily optimized to boost poxvirus protein production. A 5'-poly(A leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5'- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5'-poly(A leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES. These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

    14. High-resolution pyrimidine- and ribose-specific 4D HCCH-COSY spectra of RNA using the filter diagonalization method

      International Nuclear Information System (INIS)

      Douglas, Justin T.; Latham, Michael P.; Armstrong, Geoffrey S.; Bendiak, Brad; Pardi, Arthur

      2008-01-01

      The NMR spectra of nucleic acids suffer from severe peak overlap, which complicates resonance assignments. 4D NMR experiments can overcome much of the degeneracy in 2D and 3D spectra; however, the linear increase in acquisition time with each new dimension makes it impractical to acquire high-resolution 4D spectra using standard Fourier transform (FT) techniques. The filter diagonalization method (FDM) is a numerically efficient algorithm that fits the entire multi-dimensional time-domain data to a set of multi-dimensional oscillators. Selective 4D constant-time HCCH-COSY experiments that correlate the H5-C5-C6-H6 base spin systems of pyrimidines or the H1'-C1'-C2'-H2' spin systems of ribose sugars were acquired on the 13 C-labeled iron responsive element (IRE) RNA. FDM-processing of these 4D experiments recorded with only 8 complex points in the indirect dimensions showed superior spectral resolution than FT-processed spectra. Practical aspects of obtaining optimal FDM-processed spectra are discussed. The results here demonstrate that FDM-processing can be used to obtain high-resolution 4D spectra on a medium sized RNA in a fraction of the acquisition time normally required for high-resolution, high-dimensional spectra

    15. Generation of miRNA sponge constructs

      NARCIS (Netherlands)

      Kluiver, Joost; Slezak-Prochazka, Izabella; Smigielska-Czepiel, Katarzyna; Halsema, Nancy; Kroesen, Bart-Jan; van den Berg, Anke

      2012-01-01

      MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We

    16. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein

      OpenAIRE

      Pinder, Benjamin D; Smibert, Craig A

      2012-01-01

      Argonaute 1 directly interacts with the RNA binding protein Smaug in Drosophila, is thereby recruited to the Smaug target nanos mRNA and is required for Smaug-mediated translational repression of the nanos mRNA.

    17. IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions.

      Science.gov (United States)

      Mann, Martin; Wright, Patrick R; Backofen, Rolf

      2017-07-03

      The IntaRNA algorithm enables fast and accurate prediction of RNA-RNA hybrids by incorporating seed constraints and interaction site accessibility. Here, we introduce IntaRNAv2, which enables enhanced parameterization as well as fully customizable control over the prediction modes and output formats. Based on up to date benchmark data, the enhanced predictive quality is shown and further improvements due to more restrictive seed constraints are highlighted. The extended web interface provides visualizations of the new minimal energy profiles for RNA-RNA interactions. These allow a detailed investigation of interaction alternatives and can reveal potential interaction site multiplicity. IntaRNAv2 is freely available (source and binary), and distributed via the conda package manager. Furthermore, it has been included into the Galaxy workflow framework and its already established web interface enables ad hoc usage. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

    18. Direct, rapid RNA sequence analysis

      International Nuclear Information System (INIS)

      Peattie, D.A.

      1987-01-01

      The original methods of RNA sequence analysis were based on enzymatic production and chromatographic separation of overlapping oligonucleotide fragments from within an RNA molecule followed by identification of the mononucleotides comprising the oligomer. Over the past decade the field of nucleic acid sequencing has changed dramatically, however, and RNA molecules now can be sequenced in a variety of more streamlined fashions. Most of the more recent advances in RNA sequencing have involved one-dimensional electrophoretic separation of 32 P-end-labeled oligoribonucleotides on polyacrylamide gels. In this chapter the author discusses two of these methods for determining the nucleotide sequences of RNA molecules rapidly: the chemical method and the enzymatic method. Both methods are direct and degradative, i.e., they rely on fragmatic and chemical approaches should be utilized. The single-strand-specific ribonucleases (A, T 1 , T 2 , and S 1 ) provide an efficient means to locate double-helical regions rapidly, and the chemical reactions provide a means to determine the RNA sequence within these regions. In addition, the chemical reactions allow one to assign interactions to specific atoms and to distinguish secondary interactions from tertiary ones. If the RNA molecule is small enough to be sequenced directly by the enzymatic or chemical method, the probing reactions can be done easily at the same time as sequencing reactions

    19. Cofactors in the RNA World

      Science.gov (United States)

      Ditzler, Mark A.

      2014-01-01

      RNA world theories figure prominently in many scenarios for the origin and early evolution of life. These theories posit that RNA molecules played a much larger role in ancient biology than they do now, acting both as the dominant biocatalysts and as the repository of genetic information. Many features of modern RNA biology are potential examples of molecular fossils from an RNA world, such as the pervasive involvement of nucleotides in coenzymes, the existence of natural aptamers that bind these coenzymes, the existence of natural ribozymes, a biosynthetic pathway in which deoxynucleotides are produced from ribonucleotides, and the central role of ribosomal RNA in protein synthesis in the peptidyl transferase center of the ribosome. Here, we uses both a top-down approach that evaluates RNA function in modern biology and a bottom-up approach that examines the capacities of RNA independent of modern biology. These complementary approaches exploit multiple in vitro evolution techniques coupled with high-throughput sequencing and bioinformatics analysis. Together these complementary approaches advance our understanding of the most primitive organisms, their early evolution, and their eventual transition to modern biochemistry.

    20. Efficient RNA structure comparison algorithms.

      Science.gov (United States)

      Arslan, Abdullah N; Anandan, Jithendar; Fry, Eric; Monschke, Keith; Ganneboina, Nitin; Bowerman, Jason

      2017-12-01

      Recently proposed relative addressing-based ([Formula: see text]) RNA secondary structure representation has important features by which an RNA structure database can be stored into a suffix array. A fast substructure search algorithm has been proposed based on binary search on this suffix array. Using this substructure search algorithm, we present a fast algorithm that finds the largest common substructure of given multiple RNA structures in [Formula: see text] format. The multiple RNA structure comparison problem is NP-hard in its general formulation. We introduced a new problem for comparing multiple RNA structures. This problem has more strict similarity definition and objective, and we propose an algorithm that solves this problem efficiently. We also develop another comparison algorithm that iteratively calls this algorithm to locate nonoverlapping large common substructures in compared RNAs. With the new resulting tools, we improved the RNASSAC website (linked from http://faculty.tamuc.edu/aarslan ). This website now also includes two drawing tools: one specialized for preparing RNA substructures that can be used as input by the search tool, and another one for automatically drawing the entire RNA structure from a given structure sequence.

    1. Inositol-Requiring Enzyme 1-Mediated Downregulation of MicroRNA (miR)-146a and miR-155 in Primary Dermal Fibroblasts across Three TNFRSF1A Mutations Results in Hyperresponsiveness to Lipopolysaccharide.

      Science.gov (United States)

      Harrison, Stephanie R; Scambler, Thomas; Oubussad, Lylia; Wong, Chi; Wittmann, Miriam; McDermott, Michael F; Savic, Sinisa

      2018-01-01

      Tumor necrosis factor (TNF)-receptor-associated periodic fever syndrome (TRAPS) is a rare monogenic autoinflammatory disorder characterized by mutations in the TNFRSF1A gene, causing TNF-receptor 1 (TNFR1) misfolding, increased cellular stress, activation of the unfolded protein response (UPR), and hyperresponsiveness to lipopolysaccharide (LPS). Both microRNA (miR)-146a and miR-155 provide negative feedback for LPS-toll-like receptor 2/4 signaling and cytokine production, through regulation of nuclear factor kappa B (NF-κB). In this study, we hypothesized that proinflammatory cytokine signaling in TRAPS downregulates these two miRs, resulting in LPS-induced hyperresponsiveness in TRAPS dermal fibroblasts (DFs), irrespective of the underlying genetic mutation. Primary DF were isolated from skin biopsies of TRAPS patients and healthy controls (HC). TNFR1 cell surface expression was measured using immunofluorescence. DF were stimulated with LPS, interleukin (IL)-1β, thapsigargin, or TNF, with and without inositol-requiring enzyme 1 (IRE1) inhibitor (4u8C), following which miR-146a and miR-155 expression was measured by RT-qPCR. IL-1β, IL-6, and TNF secretion was measured by enzyme-linked immunosorbent assays, and baseline expression of 384 different miRs was assessed using microfluidics assays. TNFR1 was found to be expressed on the surface of HC DF but expression was deficient in all samples with TRAPS-associated mutations. HC DF showed significant dose-dependent increases in both miR-146a and miR-155 expression levels in response to LPS; however, TRAPS DF failed to upregulate either miR-146a or miR-155 under the same conditions. This lack of miR-146a and miR-155 upregulation was associated with increased proinflammatory cytokine production in TRAPS DF in response to LPS challenge, which was abrogated by 4u8C. Incubation of HC DF with IL-1β led to downregulation of miR-146a and miR-155 expression, which was dependent on IRE1 enzyme. We observed global

    2. Alternative RNA splicing and cancer

      Science.gov (United States)

      Liu, Sali; Cheng, Chonghui

      2015-01-01

      Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

    3. The ViennaRNA web services.

      Science.gov (United States)

      Gruber, Andreas R; Bernhart, Stephan H; Lorenz, Ronny

      2015-01-01

      The ViennaRNA package is a widely used collection of programs for thermodynamic RNA secondary structure prediction. Over the years, many additional tools have been developed building on the core programs of the package to also address issues related to noncoding RNA detection, RNA folding kinetics, or efficient sequence design considering RNA-RNA hybridizations. The ViennaRNA web services provide easy and user-friendly web access to these tools. This chapter describes how to use this online platform to perform tasks such as prediction of minimum free energy structures, prediction of RNA-RNA hybrids, or noncoding RNA detection. The ViennaRNA web services can be used free of charge and can be accessed via http://rna.tbi.univie.ac.at.

    4. Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

      NARCIS (Netherlands)

      Kluiver, Joost; Gibcus, Johan H.; Hettinga, Chris; Adema, Annelies; Richter, Mareike K. S.; Halsema, Nancy; Slezak-Prochazka, Izabella; Ding, Ye; Kroesen, Bart-Jan; van den Berg, Anke

      2012-01-01

      MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and

    5. Russian ambassador vents ire / Milda Seputyte

      Index Scriptorium Estoniae

      Seputyte, Milda

      2005-01-01

      Vene suursaadik Leedus avaldas oma kirjas Leedu päevalehe toimetajale pahameelt, kuna ajaleht oli oma artiklis väitnud, et Venemaa välisminister plaanib suursaadikut tagasi kutsuda seoses eksitava info saamisega president Valdas Adamkuse suhtumise kohta 9. mai pidustustesse Moskvas

    6. Identification of Subtype Specific miRNA-mRNA Functional Regulatory Modules in Matched miRNA-mRNA Expression Data: Multiple Myeloma as a Case

      Directory of Open Access Journals (Sweden)

      Yunpeng Zhang

      2015-01-01

      Full Text Available Identification of miRNA-mRNA modules is an important step to elucidate their combinatorial effect on the pathogenesis and mechanisms underlying complex diseases. Current identification methods primarily are based upon miRNA-target information and matched miRNA and mRNA expression profiles. However, for heterogeneous diseases, the miRNA-mRNA regulatory mechanisms may differ between subtypes, leading to differences in clinical behavior. In order to explore the pathogenesis of each subtype, it is important to identify subtype specific miRNA-mRNA modules. In this study, we integrated the Ping-Pong algorithm and multiobjective genetic algorithm to identify subtype specific miRNA-mRNA functional regulatory modules (MFRMs through integrative analysis of three biological data sets: GO biological processes, miRNA target information, and matched miRNA and mRNA expression data. We applied our method on a heterogeneous disease, multiple myeloma (MM, to identify MM subtype specific MFRMs. The constructed miRNA-mRNA regulatory networks provide modular outlook at subtype specific miRNA-mRNA interactions. Furthermore, clustering analysis demonstrated that heterogeneous MFRMs were able to separate corresponding MM subtypes. These subtype specific MFRMs may aid in the further elucidation of the pathogenesis of each subtype and may serve to guide MM subtype diagnosis and treatment.

    7. Predicting and Modeling RNA Architecture

      Science.gov (United States)

      Westhof, Eric; Masquida, Benoît; Jossinet, Fabrice

      2011-01-01

      SUMMARY A general approach for modeling the architecture of large and structured RNA molecules is described. The method exploits the modularity and the hierarchical folding of RNA architecture that is viewed as the assembly of preformed double-stranded helices defined by Watson-Crick base pairs and RNA modules maintained by non-Watson-Crick base pairs. Despite the extensive molecular neutrality observed in RNA structures, specificity in RNA folding is achieved through global constraints like lengths of helices, coaxiality of helical stacks, and structures adopted at the junctions of helices. The Assemble integrated suite of computer tools allows for sequence and structure analysis as well as interactive modeling by homology or ab initio assembly with possibilities for fitting within electronic density maps. The local key role of non-Watson-Crick pairs guides RNA architecture formation and offers metrics for assessing the accuracy of three-dimensional models in a more useful way than usual root mean square deviation (RMSD) values. PMID:20504963

    8. Shielding the messenger (RNA): microRNA-based anticancer therapies

      Science.gov (United States)

      Sotillo, Elena; Thomas-Tikhonenko, Andrei

      2011-01-01

      It has been a decade since scientists realized that microRNAs (miRNAs) are not an oddity invented by worms to regulate gene expression at post-transcriptional levels. Rather, many of these 21–22-nucleotide-short RNAs exist in invertebrates and vertebrates alike and some of them are in fact highly conserved. miRNAs are now recognized as an important class of non-coding small RNAs that inhibit gene expression by targeting mRNA stability and translation. In the last ten years, our knowledge of the miRNAs world was expanding at vertiginous speed, propelled by the development of computational engines for miRNA identification and target prediction, biochemical tools and techniques to modulate miRNA activity, and last but not least, the emergence of miRNA-centric animal models. One important conclusion that has emerged from this effort is that many microRNAs and their cognate targets are strongly implicated in cancer, either as oncogenes or tumor and metastasis suppressors. In this review we will discuss the diverse role that miRNAs play in cancer initiation and progression and also the tools with which miRNA expression could be corrected in vivo. While the idea of targeting microRNAs towards therapeutic ends is getting considerable traction, basic, translational, and clinical research done in the next few years will tell whether this promise is well-founded. PMID:21514318

    9. Chaperoning 5S RNA assembly.

      Science.gov (United States)

      Madru, Clément; Lebaron, Simon; Blaud, Magali; Delbos, Lila; Pipoli, Juliana; Pasmant, Eric; Réty, Stéphane; Leulliot, Nicolas

      2015-07-01

      In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. © 2015 Madru et al.; Published by Cold Spring Harbor Laboratory Press.

    10. Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

      Directory of Open Access Journals (Sweden)

      Hiraku Takada

      Full Text Available Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon, within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons

    11. PHYSICS DIV. QUARTERLY REPORT, MAY, JUNE, JULY, 1950

      Energy Technology Data Exchange (ETDEWEB)

      Various

      1950-09-06

      The X-ray Spectrum Produced by 322 Mev Electrons Striking a Platinum Target. The differential energy spectrum of the photons produced by 322 Mev electrons striking a 20 mil platinum target was measured by observing the energy of 3467 pairs produced in a one mil thick lead foil in a Wilson cloud chamber in a magnetic field of 10,000 gauss. The spectrum is found to agree with that predicted by the Bethe-Heitler bremsstrahlung theory using a Thomas-Fermi model with suitable corrections for the thickness of the target. The energy of the 322 Mev electrons was determined by the spectrum of the photons observed in this experiment. Apparatus used and the experimental data and results obtained are reported fully in UCRL-660.

    12. RegRNA: an integrated web server for identifying regulatory RNA motifs and elements

      OpenAIRE

      Huang, Hsi-Yuan; Chien, Chia-Hung; Jen, Kuan-Hua; Huang, Hsien-Da

      2006-01-01

      Numerous regulatory structural motifs have been identified as playing essential roles in transcriptional and post-transcriptional regulation of gene expression. RegRNA is an integrated web server for identifying the homologs of regulatory RNA motifs and elements against an input mRNA sequence. Both sequence homologs and structural homologs of regulatory RNA motifs can be recognized. The regulatory RNA motifs supported in RegRNA are categorized into several classes: (i) motifs in mRNA 5′-untra...

    13. Analysis of intermolecular RNA-RNA recombination by rubella virus

      International Nuclear Information System (INIS)

      Adams, Sandra D.; Tzeng, W.-P.; Chen, M.-H.; Frey, Teryl K.

      2003-01-01

      To investigate whether rubella virus (RUB) undergoes intermolecular RNA-RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5'-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3'-proximal structural protein (SP) ORF, and maintained the 3' end of the genome, including the putative 3' cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3' half of the genome including the SP-ORF and 3' CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA-RNA recombination. Using transcripts tagged with a 3'-terminal deletion, it was found that recombinants contained the 3' end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3' CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3' CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3' end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating

    14. MicroRNA mimicry blocks pulmonary fibrosis

      NARCIS (Netherlands)

      Montgomery, Rusty L; Yu, Guoying; Latimer, Paul A; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

      2014-01-01

      Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular

    15. Biochemistry and Function of the RNA Exosomes

      DEFF Research Database (Denmark)

      Lubas, Michal Szymon; Chlebowski, Aleksander; Dziembowski, Andrzej

      2012-01-01

      Discovery of the evolutionary conserved RNA exosome was a milestone in RNA biology. First identified as an activity essential for the processing of ribosomal RNA, the exosome has since proved to be central for RNA processing and degradation in both the nucleus and the cytoplasm of eukaryotic cell...

    16. The crystal structure of tRNA

      Indian Academy of Sciences (India)

      Madhu

      of yeast alanine tRNA by Robert Holley's group at Cornell. University ... decode nonsense codons) with John Smith and Brenner. However, my ... tRNA from 10 g of unfractionated tRNA. ... tRNA crystals were, in fact, protein (Hendrikson et al.

    17. Salvadorianae. III. Una descripció prelinneana de Silene niceensis All., deguda a Jaume Salvador i Pedrol, a l'edició de 1686 del Botanicum Monspeliense de Pèire Magnol

      Directory of Open Access Journals (Sweden)

      Camarasa, Josep M.

      2000-12-01

      Full Text Available Silene niceensis All. (Cariophillaceae is a plant found on litoral sands of Western Mediterranian and also of Iberian Atlantic shores from Gibraltar to the Rias Baixas and in some places in Greece. It had been described twice in prelinnean times but Linné has neglected it in his works. First prelinnean description, though published by Pierre Magnol in the Appedix to the second edition (1686 of Botanicum monspeliense,has been wrote on the basis of materials and a short description sent to Magnol by Jaume Salvador i Pedrol. One specimen of this plant in Salvador Herbarium hold a short description that could be the original one by Jaume Salvador. The second one was published on a different name by Tournefort in Institutiones Rei herbariae. It is also one specimen of S. niceensisAll. in Salvador Herbarium under this identity. Pourret, studying this herbarium to put on etiquets the linnean name of each specimen (1792-98, realize the specific identity of both S. niceensis All. specimens but ignoring the description published by Allioni in 1773 he give them the new name of S. arenaria Pourr. Also Desfontaines described under the name of S. arenaria Desf. in his Flora Atlantica (1798-99 the same species on the basis of specimens collected near the Gibraltar Strait. Both. Pourrett and Desfontaines names, or today invalid and under sinonimy.

      La cariofil làcia Silene niceensis All. pròpia dels sorrats litorals de la Mediterrània occidental i present també a les costes atlàntiques ibèriques. des de l' estret de Gibraltar fins a les Rias Baixas i en alguna localitat grega, havia estar descrita al menys dues vegades temps prelinneans malgrat que Linné mateix la ignorés a les seves obres. La primera descripció prelinneana. bé que publicada por Péire Magnol a l’apèndix a la segona edició del seu Botanicum monspeliense,. es feu sobre la base de materials i fins i tot d

    18. A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

      Directory of Open Access Journals (Sweden)

      Baodong Wu

      2009-03-01

      Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

    19. Glia to axon RNA transfer.

      Science.gov (United States)

      Sotelo, José Roberto; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José Roberto; Calliari, Aldo; Cal, Karina; Bresque, Mariana; Dipaolo, Andrés; Farias, Joaquina; Mercer, John A

      2014-03-01

      The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial-to-axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024-11029; Court et al. [2011]: Glia 59:1529-1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased. Copyright © 2013 Wiley Periodicals, Inc.

    20. On topological RNA interaction structures.

      Science.gov (United States)

      Qin, Jing; Reidys, Christian M

      2013-07-01

      Recently a folding algorithm of topological RNA pseudoknot structures was presented in Reidys et al. (2011). This algorithm folds single-stranded γ-structures, that is, RNA structures composed by distinct motifs of bounded topological genus. In this article, we set the theoretical foundations for the folding of the two backbone analogues of γ structures: the RNA γ-interaction structures. These are RNA-RNA interaction structures that are constructed by a finite number of building blocks over two backbones having genus at most γ. Combinatorial properties of γ-interaction structures are of practical interest since they have direct implications for the folding of topological interaction structures. We compute the generating function of γ-interaction structures and show that it is algebraic, which implies that the numbers of interaction structures can be computed recursively. We obtain simple asymptotic formulas for 0- and 1-interaction structures. The simplest class of interaction structures are the 0-interaction structures, which represent the two backbone analogues of secondary structures.

    1. Tapping the RNA world for therapeutics.

      Science.gov (United States)

      Lieberman, Judy

      2018-04-16

      A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.

    2. RNA-Based Vaccines in Cancer Immunotherapy

      Directory of Open Access Journals (Sweden)

      Megan A. McNamara

      2015-01-01

      Full Text Available RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

    3. A Regulatory RNA Inducing Transgenerationally Inherited Phenotypes

      DEFF Research Database (Denmark)

      Jensen, Lea Møller

      . The variation in Arabidopsis enables different regulatory networks and mechanisms to shape the phenotypic characteristics. The thesis describes the identification of regulatory RNA encoded by an enzyme encoding gene. The RNA regulates by inducing transgenerationally inherited phenotypes. The function of the RNA...... is dependent on the genetic background illustrating that polymorphisms are found in either interactors or target genes of the RNA. Furthermore, the RNA provides a mechanistic link between accumulation of glucosinolate and onset of flowering....

    4. Screening of Modified RNA duplexes

      DEFF Research Database (Denmark)

      Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

      protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form......Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...

    5. TargetRNA: a tool for predicting targets of small RNA action in bacteria

      OpenAIRE

      Tjaden, Brian

      2008-01-01

      Many small RNA (sRNA) genes in bacteria act as posttranscriptional regulators of target messenger RNAs. Here, we present TargetRNA, a web tool for predicting mRNA targets of sRNA action in bacteria. TargetRNA takes as input a genomic sequence that may correspond to an sRNA gene. TargetRNA then uses a dynamic programming algorithm to search each annotated message in a specified genome for mRNAs that evince basepair-binding potential to the input sRNA sequence. Based on the calculated basepair-...

    6. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

      NARCIS (Netherlands)

      Dorssers, L.

      1983-01-01

      The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.

      Previously, an RNA-dependent RNA polymerase produced upon infection of

    7. RNA Study Using DNA Nanotechnology.

      Science.gov (United States)

      Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

      2016-01-01

      Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. Copyright © 2016 Elsevier Inc. All rights reserved.

    8. Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

      DEFF Research Database (Denmark)

      Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

      2007-01-01

      We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

    9. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein.

      Science.gov (United States)

      Pinder, Benjamin D; Smibert, Craig A

      2013-01-01

      Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

    10. Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase.

      Science.gov (United States)

      Heck, Julie A; Meng, Xiao; Frick, David N

      2009-04-01

      Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111-22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidyl-prolyl isomerase activity, is not required for stimulation of RNA synthesis.

    11. Identification of new TSGA10 transcript variants in human testis with conserved regulatory RNA elements in 5'untranslated region and distinct expression in breast cancer.

      Science.gov (United States)

      Salehipour, Pouya; Nematzadeh, Mahsa; Mobasheri, Maryam Beigom; Afsharpad, Mandana; Mansouri, Kamran; Modarressi, Mohammad Hossein

      2017-09-01

      Testis specific gene antigen 10 (TSGA10) is a cancer testis antigen involved in the process of spermatogenesis. TSGA10 could also play an important role in the inhibition of angiogenesis by preventing nuclear localization of HIF-1α. Although it has been shown that TSGA10 messenger RNA (mRNA) is mainly expressed in testis and some tumors, the transcription pattern and regulatory mechanisms of this gene remain largely unknown. Here, we report that human TSGA10 comprises at least 22 exons and generates four different transcript variants. It was identified that using two distinct promoters and splicing of exons 4 and 7 produced these transcript variants, which have the same coding sequence, but the sequence of 5'untanslated region (5'UTR) is different between them. This is significant because conserved regulatory RNA elements like upstream open reading frame (uORF) and putative internal ribosome entry site (IRES) were found in this region which have different combinations in each transcript variant and it may influence translational efficiency of them in normal or unusual environmental conditions like hypoxia. To indicate the transcription pattern of TSGA10 in breast cancer, expression of identified transcript variants was analyzed in 62 breast cancer samples. We found that TSGA10 tends to express variants with shorter 5'UTR and fewer uORF elements in breast cancer tissues. Our study demonstrates for the first time the expression of different TSGA10 transcript variants in testis and breast cancer tissues and provides a first clue to a role of TSGA10 5'UTR in regulation of translation in unusual environmental conditions like hypoxia. Copyright © 2017. Published by Elsevier B.V.

    12. Fenomenología del divorcio (o la esencia de la separación en mujeres Fenomenologia do divórcio (ou a essência da separação em mulheres Phenomenology of divorce (or the essence of separation in women

      Directory of Open Access Journals (Sweden)

      Javier Andrés Gómez-Díaz

      2011-08-01

      Full Text Available La experiencia de la separación y el divorcio en parejas heterosexuales suele tener significados diferentes asociadas a lo vivido antes, durante o después del hecho. Sin embargo, es posible hallar ciertas similitudes, así como significados emergentes que se posibilitan extraer al usar la fenomenología como un método. De un grupo de mujeres divorciadas/separadas, se extrajo información acerca de sus experiencias sobre éste fenómeno, con el propósito de comprender los sentimientos y emociones vivenciados por ellas y como medio terapéutico de significación de un hecho doloroso. Esto permitió comparar lo propuesto por fenomenólogos acerca de la separación emocional con lo vivenciado por las participantes de esta investigación.A experiência de separação e divórcio em casais heterossexuais muitas vezes tem significados diferentes associados com a vida antes, durante ou após o fato. No entanto, é possível encontrar semelhanças e significados diferentes que são possíveis de extrair usando a fenomenologia como um método. Em um grupo separado de mulheres divorciadas, foram obtidas informações sobre as suas experiências sobre esse fenômeno, a fim de compreender os sentimentos e emoções vividos por elas e como um meio terapêutico de significação de um fato doloroso. Isso permitiu comparar propostas dos fenomenólogos sobre a separação de afetividade com a experiência vivida pelas participantes deste estudo.The experience of separation and divorce in heterosexual couples often has different meanings associated to the lived fact before, during, and after it. Anyway, it is possible to find some similarities, as well as emergent meanings that are possible to extract from phenomenology as a method. From a group of separated/divorced women, information were extracted about their experience on this phenomenon, in order to understand the feelings and emotions lived by them and as a therapeutic significance of a painful episode

    13. Role of CBCA in RNA biogenesis

      DEFF Research Database (Denmark)

      Iasillo, Claudia

      RNA transcription and RNA processing are key steps in eukaryotic gene expression, which includes, therefore, RNA synthesis by RNA polymerase enzymes and a range of modifications of the pre-mRNA before the transcript can leave the nucleus and reach the cytoplasm for translation. Interestingly......, a large body of evidence suggests that these RNA processing events occur often already during transcription. One of these modifications, the co-transcriptional 5’ end capping of a nascent RNA, is occurring specifically during RNA polymerase II (RNAPII) transcription. The 5’ cap exerts its role via...... the nuclear Cap Binding Complex (CBC). This thesis focuses on the protein ARS2, which binds the CBC to form the CBCA complex. CBCA can further associate with different proteins playing different roles in RNA metabolism. For example, CBCA binds the Nuclear Exosome Targeting Complex (NEXT), which...

    14. Hydration dependent dynamics in RNA

      International Nuclear Information System (INIS)

      Olsen, Greg L.; Bardaro, Michael F.; Echodu, Dorothy C.; Drobny, Gary P.; Varani, Gabriele

      2009-01-01

      The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms-ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in 2 H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state 13 C relaxation measurements, we establish that ns-μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration

    15. RNA Editing in Plant Mitochondria

      Science.gov (United States)

      Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

      1989-12-01

      Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

    16. Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

      OpenAIRE

      Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

      2003-01-01

      For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...

    17. Modular arrangement of regulatory RNA elements.

      Science.gov (United States)

      Roßmanith, Johanna; Narberhaus, Franz

      2017-03-04

      Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.

    18. MicroRNA Delivery for Regenerative Medicine

      OpenAIRE

      Peng, Bo; Chen, Yongming; Leong, Kam W.

      2015-01-01

      MicroRNA (miRNA) directs post-transcriptional regulation of a network of genes by targeting mRNA. Although relatively recent in development, many miRNAs direct differentiation of various stem cells including induced pluripotent stem cells (iPSCs), a major player in regenerative medicine. An effective and safe delivery of miRNA holds the key to translating miRNA technologies. Both viral and nonviral delivery systems have seen success in miRNA delivery, and each approach possesses advantages an...

    19. Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

      International Nuclear Information System (INIS)

      Kolakofsky, Daniel; Le Mercier, Philippe; Iseni, Frederic; Garcin, Dominique

      2004-01-01

      mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in that the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template

    20. A distinct group of hepacivirus/pestivirus-like internal ribosomal entry sites in members of diverse picornavirus genera: evidence for modular exchange of functional noncoding RNA elements by recombination.

      Science.gov (United States)

      Hellen, Christopher U T; de Breyne, Sylvain

      2007-06-01

      The 5' untranslated regions (UTRs) of the RNA genomes of Flaviviridae of the Hepacivirus and Pestivirus genera contain internal ribosomal entry sites (IRESs) that are unrelated to the two principal classes of IRESs of Picornaviridae. The mechanism of translation initiation on hepacivirus/pestivirus (HP) IRESs, which involves factor-independent binding to ribosomal 40S subunits, also differs fundamentally from initiation on these picornavirus IRESs. Ribosomal binding to HP IRESs requires conserved sequences that form a pseudoknot and the adjacent IIId and IIIe domains; analogous elements do not occur in the two principal groups of picornavirus IRESs. Here, comparative sequence analysis was used to identify a subset of picornaviruses from multiple genera that contain 5' UTR sequences with significant similarities to HP IRESs. They are avian encephalomyelitis virus, duck hepatitis virus 1, duck picornavirus, porcine teschovirus, porcine enterovirus 8, Seneca Valley virus, and simian picornavirus. Their 5' UTRs are predicted to form several structures, in some of which the peripheral elements differ from the corresponding HP IRES elements but in which the core pseudoknot, domain IIId, and domain IIIe elements are all closely related. These findings suggest that HP-like IRESs have been exchanged between unrelated virus families by recombination and support the hypothesis that RNA viruses consist of modular coding and noncoding elements that can exchange and evolve independently.

    1. A Distinct Group of Hepacivirus/Pestivirus-Like Internal Ribosomal Entry Sites in Members of Diverse Picornavirus Genera: Evidence for Modular Exchange of Functional Noncoding RNA Elements by Recombination▿ †

      Science.gov (United States)

      Hellen, Christopher U. T.; de Breyne, Sylvain

      2007-01-01

      The 5′ untranslated regions (UTRs) of the RNA genomes of Flaviviridae of the Hepacivirus and Pestivirus genera contain internal ribosomal entry sites (IRESs) that are unrelated to the two principal classes of IRESs of Picornaviridae. The mechanism of translation initiation on hepacivirus/pestivirus (HP) IRESs, which involves factor-independent binding to ribosomal 40S subunits, also differs fundamentally from initiation on these picornavirus IRESs. Ribosomal binding to HP IRESs requires conserved sequences that form a pseudoknot and the adjacent IIId and IIIe domains; analogous elements do not occur in the two principal groups of picornavirus IRESs. Here, comparative sequence analysis was used to identify a subset of picornaviruses from multiple genera that contain 5′ UTR sequences with significant similarities to HP IRESs. They are avian encephalomyelitis virus, duck hepatitis virus 1, duck picornavirus, porcine teschovirus, porcine enterovirus 8, Seneca Valley virus, and simian picornavirus. Their 5′ UTRs are predicted to form several structures, in some of which the peripheral elements differ from the corresponding HP IRES elements but in which the core pseudoknot, domain IIId, and domain IIIe elements are all closely related. These findings suggest that HP-like IRESs have been exchanged between unrelated virus families by recombination and support the hypothesis that RNA viruses consist of modular coding and noncoding elements that can exchange and evolve independently. PMID:17392358

    2. iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

      Directory of Open Access Journals (Sweden)

      Jittrawan Thaiprasit

      2016-03-01

      Full Text Available RNA-RNA interactions play a crucial role in gene regulation in living organisms. They have gained increasing interest in the field of synthetic biology because of their potential applications in medicine and biotechnology. However, few novel regulators based on RNA-RNA interactions with desired structures and functions have been developed due to the challenges of developing design tools. Recently, we proposed a novel tool, called iDoDe, for designing RNA-RNA interacting sequences by first decomposing RNA structures into interacting domains and then designing each domain using a stochastic algorithm. However, iDoDe did not provide an optimal solution because it still lacks a mechanism to optimize the design. In this work, we have further developed the tool by incorporating a genetic algorithm (GA to find an RNA solution with maximized structural similarity and minimized hybridized RNA energy, and renamed the tool iDoRNA. A set of suitable parameters for the genetic algorithm were determined and found to be a weighting factor of 0.7, a crossover rate of 0.9, a mutation rate of 0.1, and the number of individuals per population set to 8. We demonstrated the performance of iDoRNA in comparison with iDoDe by using six RNA-RNA interaction models. It was found that iDoRNA could efficiently generate all models of interacting RNAs with far more accuracy and required far less computational time than iDoDe. Moreover, we compared the design performance of our tool against existing design tools using forty-four RNA-RNA interaction models. The results showed that the performance of iDoRNA is better than RiboMaker when considering the ensemble defect, the fitness score and computation time usage. However, it appears that iDoRNA is outperformed by NUPACK and RNAiFold 2.0 when considering the ensemble defect. Nevertheless, iDoRNA can still be an useful alternative tool for designing novel RNA-RNA interactions in synthetic biology research. The source code of iDoRNA

    3. Comprehensive characterization of lncRNA-mRNA related ceRNA network across 12 major cancers

      Science.gov (United States)

      Feng, Li; Li, Feng; Sun, Zeguo; Wu, Tan; Shi, Xinrui; Li, Jing; Li, Xia

      2016-01-01

      Recent studies indicate that long noncoding RNAs (lncRNAs) can act as competing endogenous RNAs (ceRNAs) to indirectly regulate mRNAs through shared microRNAs, which represents a novel layer of RNA crosstalk and plays critical roles in the development of tumor. However, the global regulation landscape and characterization of these lncRNA related ceRNA crosstalk in cancers is still largely unknown. Here, we systematically characterized the lncRNA related ceRNA interactions across 12 major cancers and the normal physiological states by integrating multidimensional molecule profiles of more than 5000 samples. Our study suggest the large difference of ceRNA regulation between normal and tumor states and the higher similarity across similar tissue origin of tumors. The ceRNA related molecules have more conserved features in tumor networks and they play critical roles in both the normal and tumorigenesis processes. Besides, lncRNAs in the pan-cancer ceRNA network may be potential biomarkers of tumor. By exploring hub lncRNAs, we found that these conserved key lncRNAs dominate variable tumor hallmark processes across pan-cancers. Network dynamic analysis highlights the critical roles of ceRNA regulation in tumorigenesis. By analyzing conserved ceRNA interactions, we found that miRNA mediate ceRNA regulation showed different patterns across pan-cancer; while analyzing the cancer specific ceRNA interactions reveal that lncRNAs synergistically regulated tumor driver genes of cancer hallmarks. Finally, we found that ceRNA modules have the potential to predict patient survival. Overall, our study systematically dissected the lncRNA related ceRNA networks in pan-cancer that shed new light on understanding the molecular mechanism of tumorigenesis. PMID:27580177

    4. Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

      Directory of Open Access Journals (Sweden)

      Darrin V. Bann

      2012-06-01

      Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

    5. How the RNA isolation method can affect microRNA microarray results

      DEFF Research Database (Denmark)

      Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas

      2011-01-01

      RNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results...... that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods.......The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform micro...

    6. Topology and prediction of RNA pseudoknots

      DEFF Research Database (Denmark)

      Reidys, Christian; Huang, Fenix; Andersen, Jørgen Ellegaard

      2011-01-01

      Motivation: Several dynamic programming algorithms for predicting RNA structures with pseudoknots have been proposed that differ dramatically from one another in the classes of structures considered. Results: Here, we use the natural topological classification of RNA structures in terms...

    7. RNA Structural Alignments, Part I

      DEFF Research Database (Denmark)

      Havgaard, Jakob Hull; Gorodkin, Jan

      2014-01-01

      Simultaneous alignment and secondary structure prediction of RNA sequences is often referred to as "RNA structural alignment." A class of the methods for structural alignment is based on the principles proposed by Sankoff more than 25 years ago. The Sankoff algorithm simultaneously folds and aligns...... is so high that it took more than a decade before the first implementation of a Sankoff style algorithm was published. However, with the faster computers available today and the improved heuristics used in the implementations the Sankoff-based methods have become practical. This chapter describes...... the methods based on the Sankoff algorithm. All the practical implementations of the algorithm use heuristics to make them run in reasonable time and memory. These heuristics are also described in this chapter....

    8. Fatgraph models of RNA structure

      Directory of Open Access Journals (Sweden)

      Huang Fenix

      2017-01-01

      Full Text Available In this review paper we discuss fatgraphs as a conceptual framework for RNA structures. We discuss various notions of coarse-grained RNA structures and relate them to fatgraphs.We motivate and discuss the main intuition behind the fatgraph model and showcase its applicability to canonical as well as noncanonical base pairs. Recent discoveries regarding novel recursions of pseudoknotted (pk configurations as well as their translation into context-free grammars for pk-structures are discussed. This is shown to allow for extending the concept of partition functions of sequences w.r.t. a fixed structure having non-crossing arcs to pk-structures. We discuss minimum free energy folding of pk-structures and combine these above results outlining how to obtain an inverse folding algorithm for PK structures.

    9. RNA

      African Journals Online (AJOL)

      SARAH

      30 nov. 2013 ... Keywords: FMNR, mode of management, re-greening, leadership, evolutionary trend. INTRODUCTION .... régénération : L'évolution de la densité des ligneux entre. 2005 et 2012 ..... la production et la qualité fourragères de la.

    10. Nonradioactive RNA mobility shift with chemiluminescent detection ...

      African Journals Online (AJOL)

      hesham

      RNA mobility shift is one among many procedures used to study RNA-protein interaction. Yet, there are some limitations for the radioactive RNA mobility shift including; 1) the risk of using radiolabeled nucleotides, 2) the long time to get the results; this could range from days to weeks, and 3) its high cost as compared to ...

    11. Optimization of chemiluminescent detection of mitochondrial RNA ...

      African Journals Online (AJOL)

      RNA mobility shift is one among many procedures used to study RNA-protein interaction. Yet, there are some limitations for the radioactive RNA mobility shift including; 1) the risk of using radiolabeled nucleotides, 2) the long time to get the results; this could range from days to weeks, and 3) its high cost as compared to ...

    12. RNA polymerase activity of Ustilago maydis virus

      Energy Technology Data Exchange (ETDEWEB)

      Yie, S.W.

      1986-01-01

      Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

    13. Analysis of RNA metabolism in fission yeast

      DEFF Research Database (Denmark)

      Wise, Jo Ann; Nielsen, Olaf

      2017-01-01

      Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles....

    14. Tospovirus : induction and suppression of RNA silencing

      NARCIS (Netherlands)

      Hedil, Marcio

      2016-01-01

      While infecting their hosts, viruses must deal with host immunity. In plants the antiviral RNA silencing pathway is an important part of plant innate immunity. Tospoviruses are segmented negative-stranded RNA viruses of plants. To counteract the antiviral RNA silencing response in plants,

    15. A Specific Hepatic Transfer RNA for Phosphoserine*

      Science.gov (United States)

      Mäenpää, Pekka H.; Bernfield, Merton R.

      1970-01-01

      Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [3H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [3H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNASer species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis. PMID:4943179

    16. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

      Directory of Open Access Journals (Sweden)

      Gayani N. P. Dedduwa-Mudalige

      2015-09-01

      Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

    17. Small catalytic RNA: Structure, function and application

      Energy Technology Data Exchange (ETDEWEB)

      Monforte, Joseph Albert [Univ. of California, Berkeley, CA (United States)

      1991-04-01

      We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 121±s are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

    18. Supplementary data: Materials and methods RNA expression ...

      Indian Academy of Sciences (India)

      ritt8

      Supplementary data: Materials and methods. RNA expression analysis. Freshly collected tissue was taken in TRIzol reagent for total RNA isolation according to the manufacturer's protocol. The cDNA synthesis was carried out in 1 μg total RNA using Random hexamer (Invitrogen, Carlsbad, USA) and Superscript III ...

    19. Regulatory RNAs derived from transfer RNA?

      Science.gov (United States)

      Pederson, Thoru

      2010-10-01

      Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.

    20. Regulatory BC1 RNA in Cognitive Control

      Science.gov (United States)

      Iacoangeli, Anna; Dosunmu, Aderemi; Eom, Taesun; Stefanov, Dimitre G.; Tiedge, Henri

      2017-01-01

      Dendritic regulatory BC1 RNA is a non-protein-coding (npc) RNA that operates in the translational control of gene expression. The absence of BC1 RNA in BC1 knockout (KO) animals causes translational dysregulation that entails neuronal phenotypic alterations including prolonged epileptiform discharges, audiogenic seizure activity in vivo, and…

    1. Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

      Energy Technology Data Exchange (ETDEWEB)

      Ishida, Hisashi; Tatsumi, Tomohide; Hosui, Atsushi; Nawa, Takatoshi; Kodama, Takahiro; Shimizu, Satoshi; Hikita, Hayato; Hiramatsu, Naoki; Kanto, Tatsuya [Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita 565-0871 (Japan); Hayashi, Norio [Kansai Rosai Hospital, 3-1-69, Inabaso, Amagasaki 660-8511 (Japan); Takehara, Tetsuo, E-mail: takehara@gh.med.osaka-u.ac.jp [Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita 565-0871 (Japan)

      2011-08-19

      Highlights: {yields} HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. {yields} Transfection of miR-192, -215, and -491 enhanced HCV replication. {yields} Transfection of miR-491 inhibited Akt phosphorylation. {yields} Akt inhibition could be responsible for augmentation of HCV replication by miR-491. -- Abstract: The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

    2. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

      DEFF Research Database (Denmark)

      Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

      2015-01-01

      ) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

    3. Effective Anti-miRNA Oligonucleotides Show High Releasing Rate of MicroRNA from RNA-Induced Silencing Complex.

      Science.gov (United States)

      Ariyoshi, Jumpei; Matsuyama, Yohei; Kobori, Akio; Murakami, Akira; Sugiyama, Hiroshi; Yamayoshi, Asako

      2017-10-01

      MicroRNAs (miRNAs) regulate gene expression by forming RNA-induced silencing complexes (RISCs) and have been considered as promising therapeutic targets. MiRNA is an essential component of RISC for the modulation of gene expression. Therefore, the release of miRNA from RISC is considered as an effective method for the inhibition of miRNA functions. In our previous study, we reported that anti-miRNA oligonucleotides (AMOs), which are composed of the 2'-O-methyl (2'-OMe) RNA, could induce the release of miRNA from RISC. However, the mechanisms underlying the miRNA-releasing effects of chemically modified AMOs, which are conventionally used as anti-cancer drugs, are still unclear. In this study, we investigated the relationship between the miRNA releasing rate from RISC and the inhibitory effect on RISC activity (IC 50 ) using conventional chemically modified AMOs. We demonstrated that the miRNA-releasing effects of AMOs are directly proportional to the IC 50 values, and AMOs, which have an ability to promote the release of miRNA from RISC, can effectively inhibit RISC activity in living cells.

    4. Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

      Science.gov (United States)

      Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

      2010-11-01

      The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

    5. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

      Science.gov (United States)

      Goldfarb, Katherine C; Cech, Thomas R

      2017-01-01

      MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

    6. The early history of tRNA recognition by aminoacyl-tRNA synthetases

      Indian Academy of Sciences (India)

      Madhu

      2006-10-04

      Oct 4, 2006 ... Discovery of aminoacyl-tRNA synthetases and importance ... The pioneering work of Fritz Lipmann on the high-energy ... the peculiar structural and functional relationships tRNAs ... a bulk of only 20 families of tRNA molecules in contrast ...... balance of tRNA and aminoacyl-tRNA synthetase; Science 242.

    7. O perfil de casais que vivenciam divórcios consensuais e litigiosos: uma análise das demandas judiciais El perfil de las parejas que experimentan divorcios consensuales y litigiosos: un análisis de los litigios The profile of couples who experience consensual divorces and litigation: an analysis of lawsuits

      Directory of Open Access Journals (Sweden)

      Eliana Piccoli Zordan

      2012-08-01

      Full Text Available Este estudo objetivou identificar os motivos, circunstâncias e desdobramentos das separações conjugais com base em registros em processos judiciais. Realizou-se uma análise documental de 152 separações conjugais arquivadas de 1992 a 2006, no Fórum de uma cidade do interior do RS. Fez-se uma análise descritiva e discriminante dos dados. Os motivos mais citados referem-se ao conflito conjugal em seus diferentes matizes e intensidades. Quanto ao desfecho, constatou-se que 46,7% dos processos foram consensuais, 30,9% litigiosos e em 22,4% houve abandono da ação ou reconciliação. O perfil dos casais discrimina-se prioritariamente pelo nível socioeconômico no que diz respeito aos processos consensuais ou litigiosos. Separações consensuais são mais comuns em níveis socioeconómicos baixos e litigiosas em altos. Os dados indicam que o divórcio pode ser considerado uma crise vital contemporânea e demanda dos profissionais maior complexidade nas suas intervenções tanto de ordem jurídica como psicológica no fenómeno.Este estudio tuvo como objetivo identificar las razones, circunstancias y consecuencias de las separaciones matrimoniales por medio de registros en procesos judiciales. Se realizó un análisis documental de 152 separaciones conyugales archivadas desde 1992 hasta 2006, en el Foro de una ciudad de RS-Brasil. Se hizo un análisis descriptivo y discriminante de datos. Los motivos más citados se refieren a conflictos de pareja en sus diferentes matices e intensidades. En cuanto a los resultados, se encontró que el 46,7% de los casos fueron consensuales, el 30,9% litigiosos, y en el 22,4% de ellos hubo abandono de la acción o reconciliación. El perfil de las parejas es discriminado principalmente por el nivel socioeconómico en relación a los procesos consensuales o litigiosos. Separaciones consensuales son más frecuentes en niveles socioeconómicos más bajos y litigiosas en las altas. Los datos indican que el

    8. Cooperation of an RNA Packaging Signal and a Viral Envelope Protein in Coronavirus RNA Packaging

      OpenAIRE

      Narayanan, Krishna; Makino, Shinji

      2001-01-01

      Murine coronavirus mouse hepatitis virus (MHV) produces a genome-length mRNA, mRNA 1, and six or seven species of subgenomic mRNAs in infected cells. Among these mRNAs, only mRNA 1 is efficiently packaged into MHV particles. MHV N protein binds to all MHV mRNAs, whereas envelope M protein interacts only with mRNA 1. This M protein-mRNA 1 interaction most probably determines the selective packaging of mRNA 1 into MHV particles. A short cis-acting MHV RNA packaging signal is necessary and suffi...

    9. Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA

      Science.gov (United States)

      Zhu, Yanglong; Stribinskis, Vilius; Ramos, Kenneth S.; Li, Yong

      2006-01-01

      RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5′ termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution. PMID:16540690

    10. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

      International Nuclear Information System (INIS)

      Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

      2006-01-01

      RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

    11. Using RNA Interference to Study Protein Function

      OpenAIRE

      Curtis, Carol D.; Nardulli, Ann M.

      2009-01-01

      RNA interference can be extremely useful in determining the function of an endogenously-expressed protein in its normal cellular environment. In this chapter, we describe a method that uses small interfering RNA (siRNA) to knock down mRNA and protein expression in cultured cells so that the effect of a putative regulatory protein on gene expression can be delineated. Methods of assessing the effectiveness of the siRNA procedure using real time quantitative PCR and Western analysis are also in...

    12. Analysis of extracellular RNA by digital PCR

      Directory of Open Access Journals (Sweden)

      Kenji eTakahashi

      2014-06-01

      Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

    13. Characteristics and Prediction of RNA Structure

      Directory of Open Access Journals (Sweden)

      Hengwu Li

      2014-01-01

      Full Text Available RNA secondary structures with pseudoknots are often predicted by minimizing free energy, which is NP-hard. Most RNAs fold during transcription from DNA into RNA through a hierarchical pathway wherein secondary structures form prior to tertiary structures. Real RNA secondary structures often have local instead of global optimization because of kinetic reasons. The performance of RNA structure prediction may be improved by considering dynamic and hierarchical folding mechanisms. This study is a novel report on RNA folding that accords with the golden mean characteristic based on the statistical analysis of the real RNA secondary structures of all 480 sequences from RNA STRAND, which are validated by NMR or X-ray. The length ratios of domains in these sequences are approximately 0.382L, 0.5L, 0.618L, and L, where L is the sequence length. These points are just the important golden sections of sequence. With this characteristic, an algorithm is designed to predict RNA hierarchical structures and simulate RNA folding by dynamically folding RNA structures according to the above golden section points. The sensitivity and number of predicted pseudoknots of our algorithm are better than those of the Mfold, HotKnots, McQfold, ProbKnot, and Lhw-Zhu algorithms. Experimental results reflect the folding rules of RNA from a new angle that is close to natural folding.

    14. siRNA and innate immunity.

      Science.gov (United States)

      Robbins, Marjorie; Judge, Adam; MacLachlan, Ian

      2009-06-01

      Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. Synthetic siRNA in delivery vehicles that facilitate cellular uptake can induce high levels of inflammatory cytokines and interferons after systemic administration in mammals and in primary human blood cell cultures. This activation is predominantly mediated by immune cells, normally via a Toll-like receptor (TLR) pathway. The siRNA sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively nonimmune cell activation may also occur, typically resulting from siRNA interaction with cytoplasmic RNA sensors such as RIG1. As immune activation by siRNA-based drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans, understanding and abrogating this activity will be a critical component in the development of safe and effective therapeutics. This review describes the intracellular mechanisms of innate immune activation by siRNA, the design of appropriate sequences and chemical modification approaches, and suitable experimental methods for studying their effects, with a view toward reducing siRNA-mediated off-target effects.

    15. TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

      Science.gov (United States)

      Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

    16. MysiRNA-designer: a workflow for efficient siRNA design.

      Directory of Open Access Journals (Sweden)

      Mohamed Mysara

      Full Text Available The design of small interfering RNA (siRNA is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.

    17. 5S rRNA and ribosome.

      Science.gov (United States)

      Gongadze, G M

      2011-12-01

      5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

    18. Kin Selection in the RNA World.

      Science.gov (United States)

      Levin, Samuel R; West, Stuart A

      2017-12-05

      Various steps in the RNA world required cooperation. Why did life's first inhabitants, from polymerases to synthetases, cooperate? We develop kin selection models of the RNA world to answer these questions. We develop a very simple model of RNA cooperation and then elaborate it to model three relevant issues in RNA biology: (1) whether cooperative RNAs receive the benefits of cooperation; (2) the scale of competition in RNA populations; and (3) explicit replicator diffusion and survival. We show: (1) that RNAs are likely to express partial cooperation; (2) that RNAs will need mechanisms for overcoming local competition; and (3) in a specific example of RNA cooperation, persistence after replication and offspring diffusion allow for cooperation to overcome competition. More generally, we show how kin selection can unify previously disparate answers to the question of RNA world cooperation.

    19. MicroRNA and cancer

      DEFF Research Database (Denmark)

      Jansson, Martin D; Lund, Anders H

      2012-01-01

      biological phenomena and pathologies. The best characterized non-coding RNA family consists in humans of about 1400 microRNAs for which abundant evidence have demonstrated fundamental importance in normal development, differentiation, growth control and in human diseases such as cancer. In this review, we...... summarize the current knowledge and concepts concerning the involvement of microRNAs in cancer, which have emerged from the study of cell culture and animal model systems, including the regulation of key cancer-related pathways, such as cell cycle control and the DNA damage response. Importantly, micro...

    20. RNA2DMut: a web tool for the design and analysis of RNA structure mutations.

      Science.gov (United States)

      Moss, Walter N

      2018-03-01

      With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis - and trans -regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/. © 2018 Moss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

    1. RNA versatility, flexibility, and thermostability for practice in RNA nanotechnology and biomedical applications.

      Science.gov (United States)

      Haque, Farzin; Pi, Fengmei; Zhao, Zhengyi; Gu, Shanqing; Hu, Haibo; Yu, Hang; Guo, Peixuan

      2018-01-01

      In recent years, RNA has attracted widespread attention as a unique biomaterial with distinct biophysical properties for designing sophisticated architectures in the nanometer scale. RNA is much more versatile in structure and function with higher thermodynamic stability compared to its nucleic acid counterpart DNA. Larger RNA molecules can be viewed as a modular structure built from a combination of many 'Lego' building blocks connected via different linker sequences. By exploiting the diversity of RNA motifs and flexibility of structure, varieties of RNA architectures can be fabricated with precise control of shape, size, and stoichiometry. Many structural motifs have been discovered and characterized over the years and the crystal structures of many of these motifs are available for nanoparticle construction. For example, using the flexibility and versatility of RNA structure, RNA triangles, squares, pentagons, and hexagons can be constructed from phi29 pRNA three-way-junction (3WJ) building block. This review will focus on 2D RNA triangles, squares, and hexamers; 3D and 4D structures built from basic RNA building blocks; and their prospective applications in vivo as imaging or therapeutic agents via specific delivery and targeting. Methods for intracellular cloning and expression of RNA molecules and the in vivo assembly of RNA nanoparticles will also be reviewed. WIREs RNA 2018, 9:e1452. doi: 10.1002/wrna.1452 This article is categorized under: RNA Methods > RNA Nanotechnology RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs. © 2017 Wiley Periodicals, Inc.

    2. Evaluation of microRNA alignment techniques

      Science.gov (United States)

      Kaspi, Antony; El-Osta, Assam

      2016-01-01

      Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing. PMID:27284164

    3. MicroRNA mimicry blocks pulmonary fibrosis

      Science.gov (United States)

      Montgomery, Rusty L; Yu, Guoying; Latimer, Paul A; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

      2014-01-01

      Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis. PMID:25239947

    4. Modulation of RNA function by aminoglycoside antibiotics.

      Science.gov (United States)

      Schroeder, R; Waldsich, C; Wank, H

      2000-01-04

      One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

    5. Movement of regulatory RNA between animal cells.

      Science.gov (United States)

      Jose, Antony M

      2015-07-01

      Recent studies suggest that RNA can move from one cell to another and regulate genes through specific base-pairing. Mechanisms that modify or select RNA for secretion from a cell are unclear. Secreted RNA can be stable enough to be detected in the extracellular environment and can enter the cytosol of distant cells to regulate genes. Mechanisms that import RNA into the cytosol of an animal cell can enable uptake of RNA from many sources including other organisms. This role of RNA is akin to that of steroid hormones, which cross cell membranes to regulate genes. The potential diagnostic use of RNA in human extracellular fluids has ignited interest in understanding mechanisms that enable the movement of RNA between animal cells. Genetic model systems will be essential to gain more confidence in proposed mechanisms of RNA transport and to connect an extracellular RNA with a specific biological function. Studies in the worm C. elegans and in other animals have begun to reveal parts of this novel mechanism of cell-to-cell communication. Here, I summarize the current state of this nascent field, highlight the many unknowns, and suggest future directions. © 2015 Wiley Periodicals, Inc.

    6. Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

      Science.gov (United States)

      Martos, Laura; Fernández-Pardo, Álvaro; Oto, Julia; Medina, Pilar; España, Francisco; Navarro, Silvia

      2017-01-01

      microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. PMID:29077772

    7. Henipavirus RNA in African bats.

      Directory of Open Access Journals (Sweden)

      Jan Felix Drexler

      Full Text Available BACKGROUND: Henipaviruses (Hendra and Nipah virus are highly pathogenic members of the family Paramyxoviridae. Fruit-eating bats of the Pteropus genus have been suggested as their natural reservoir. Human Henipavirus infections have been reported in a region extending from Australia via Malaysia into Bangladesh, compatible with the geographic range of Pteropus. These bats do not occur in continental Africa, but a whole range of other fruit bats is encountered. One of the most abundant is Eidolon helvum, the African Straw-coloured fruit bat. METHODOLOGY/PRINCIPAL FINDINGS: Feces from E. helvum roosting in an urban setting in Kumasi/Ghana were tested for Henipavirus RNA. Sequences of three novel viruses in phylogenetic relationship to known Henipaviruses were detected. Virus RNA concentrations in feces were low. CONCLUSIONS/SIGNIFICANCE: The finding of novel putative Henipaviruses outside Australia and Asia contributes a significant extension of the region of potential endemicity of one of the most pathogenic virus genera known in humans.

    8. REDIdb: the RNA editing database.

      Science.gov (United States)

      Picardi, Ernesto; Regina, Teresa Maria Rosaria; Brennicke, Axel; Quagliariello, Carla

      2007-01-01

      The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at http://biologia.unical.it/py_script/search.html.

    9. 5S rRNA-derived and tRNA-derived SINEs in fruit bats.

      Science.gov (United States)

      Gogolevsky, Konstantin P; Vassetzky, Nikita S; Kramerov, Dmitri A

      2009-05-01

      Most short retroposons (SINEs) descend from cellular tRNA of 7SL RNA. Here, four new SINEs were found in megabats (Megachiroptera) but neither in microbats nor in other mammals. Two of them, MEG-RS and MEG-RL, descend from another cellular RNA, 5S rRNA; one (MEG-T2) is a tRNA-derived SINE; and MEG-TR is a hybrid tRNA/5S rRNA SINE. Insertion locus analysis suggests that these SINEs were active in the recent fruit bat evolution. Analysis of MEG-RS and MEG-RL in comparison with other few 5S rRNA-derived SINEs demonstrates that the internal RNA polymerase III promoter is their most invariant region, while the secondary structure is more variable. The mechanisms underlying the modular structure of these and other SINEs as well as their variation are discussed. The scenario of evolution of MEG SINEs is proposed.

    10. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

      KAUST Repository

      Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

      2017-01-01

      RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

    11. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

      KAUST Repository

      Köster, Tino

      2017-04-13

      RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

    12. Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

      Science.gov (United States)

      Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

      2014-05-01

      RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

    13. Role of RNase MRP in viral RNA degradation and RNA recombination.

      Science.gov (United States)

      Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

      2011-01-01

      RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

    14. Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

      Science.gov (United States)

      de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

      2016-01-01

      Abstract Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

    15. The use of 125iodine-labeled RNA for detection of the RNA binding to ribosomes

      International Nuclear Information System (INIS)

      Mori, Tomohiko; Fukuda, Mitsuru

      1975-01-01

      The in vitro labeling of RNA with radioactive iodine is the efficient method to obtain the RNA with high specific activity. The present paper reports on the application of this technique to the production of iodine-labeled RNA for use in the experiment of binding RNA to ribosomes. Tobacco mosaic virus (TMV) RNA was used as natural mRNA, and E. coli S-30 preparation was used as a source of ribosomes. The TMV-RNA was prepared by bentonite-phenol extraction from TMV, and the method used for the iodation of RNA was based on the procedure described by Getz et al. The iodine-labeled RNA was incubated in a cell-free protein synthesizing system (S-30) prepared from E. coli K-12. After the incubation, the reaction mixture was layered onto sucrose gradient, centrifuged, and fractionated into 18 fractions. Optical density at 260 nm was measured, and radioactivity was counted, for each fraction. The binding of mRNA to ribosomes occurred even at 0 deg C, and the occurrence of the nonspecific binding was also shown. Consequently, the specific binding, i.e. the formation of the initiation complex being involved in amino acid incorporation, may be estimated by subtracting the radioactivity associated with monosomes in the presence of both rRNA and ATA from that in the presence of rRNA only. It was shown that the iodine-labeled RNA can be used for the studies of binding RNA to ribosomes. (Kako, I.)

    16. Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

      Science.gov (United States)

      Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

      2014-02-04

      TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

    17. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

      Science.gov (United States)

      Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

      2015-12-01

      Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

    18. Argonaute: The executor of small RNA function.

      Science.gov (United States)

      Azlan, Azali; Dzaki, Najat; Azzam, Ghows

      2016-08-20

      The discovery of small non-coding RNAs - microRNA (miRNA), short interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) - represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation. In order to execute their functions, these small RNAs require physical interactions with their protein partners, the Argonaute (AGO) family proteins. Over the years, numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms. In this review, we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies, humans and other model organisms as compliment. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

    19. Designing synthetic RNA for delivery by nanoparticles

      International Nuclear Information System (INIS)

      Jedrzejczyk, Dominika; Pawlowska, Roza; Chworos, Arkadiusz; Gendaszewska-Darmach, Edyta

      2017-01-01

      The rapid development of synthetic biology and nanobiotechnology has led to the construction of various synthetic RNA nanoparticles of different functionalities and potential applications. As they occur naturally, nucleic acids are an attractive construction material for biocompatible nanoscaffold and nanomachine design. In this review, we provide an overview of the types of RNA and nucleic acid’s nanoparticle design, with the focus on relevant nanostructures utilized for gene-expression regulation in cellular models. Structural analysis and modeling is addressed along with the tools available for RNA structural prediction. The functionalization of RNA-based nanoparticles leading to prospective applications of such constructs in potential therapies is shown. The route from the nanoparticle design and modeling through synthesis and functionalization to cellular application is also described. For a better understanding of the fate of targeted RNA after delivery, an overview of RNA processing inside the cell is also provided. (topical review)

    20. Predicting RNA Structure Using Mutual Information

      DEFF Research Database (Denmark)

      Freyhult, E.; Moulton, V.; Gardner, P. P.

      2005-01-01

      , to display and predict conserved RNA secondary structure (including pseudoknots) from an alignment. Results: We show that MIfold can be used to predict simple pseudoknots, and that the performance can be adjusted to make it either more sensitive or more selective. We also demonstrate that the overall...... package. Conclusion: MIfold provides a useful supplementary tool to programs such as RNA Structure Logo, RNAalifold and COVE, and should be useful for automatically generating structural predictions for databases such as Rfam. Availability: MIfold is freely available from http......Background: With the ever-increasing number of sequenced RNAs and the establishment of new RNA databases, such as the Comparative RNA Web Site and Rfam, there is a growing need for accurately and automatically predicting RNA structures from multiple alignments. Since RNA secondary structure...

    1. Preparation of Total RNA from Fission Yeast.

      Science.gov (United States)

      Bähler, Jürg; Wise, Jo Ann

      2017-04-03

      Treatment with hot phenol breaks open fission yeast cells and begins to strip away bound proteins from RNA. Deproteinization is completed by multiple extractions with chloroform/isoamyl alcohol and separation of the aqueous and organic phases using MaXtract gel, an inert material that acts as a physical barrier between the phases. The final step is concentration of the RNA by ethanol precipitation. The protocol can be used to prepare RNA from several cultures grown in parallel, but it is important not to process too many samples at once because delays can be detrimental to RNA quality. A reasonable number of samples to process at once would be three to four for microarray or RNA sequencing analyses and six for preliminary investigations of mutants implicated in RNA metabolism. © 2017 Cold Spring Harbor Laboratory Press.

    2. A probabilistic model of RNA conformational space

      DEFF Research Database (Denmark)

      Frellsen, Jes; Moltke, Ida; Thiim, Martin

      2009-01-01

      efficient sampling of RNA conformations in continuous space, and with associated probabilities. We show that the model captures several key features of RNA structure, such as its rotameric nature and the distribution of the helix lengths. Furthermore, the model readily generates native-like 3-D......, the discrete nature of the fragments necessitates the use of carefully tuned, unphysical energy functions, and their non-probabilistic nature impairs unbiased sampling. We offer a solution to the sampling problem that removes these important limitations: a probabilistic model of RNA structure that allows......The increasing importance of non-coding RNA in biology and medicine has led to a growing interest in the problem of RNA 3-D structure prediction. As is the case for proteins, RNA 3-D structure prediction methods require two key ingredients: an accurate energy function and a conformational sampling...

    3. RNA-Binding Proteins in Plant Immunity

      Directory of Open Access Journals (Sweden)

      Virginia Woloshen

      2011-01-01

      Full Text Available Plant defence responses against pathogen infection are crucial to plant survival. The high degree of regulation of plant immunity occurs both transcriptionally and posttranscriptionally. Once transcribed, target gene RNA must be processed prior to translation. This includes polyadenylation, 5′capping, editing, splicing, and mRNA export. RNA-binding proteins (RBPs have been implicated at each level of RNA processing. Previous research has primarily focused on structural RNA-binding proteins of yeast and mammals; however, more recent work has characterized a number of plant RBPs and revealed their roles in plant immune responses. This paper provides an update on the known functions of RBPs in plant immune response regulation. Future in-depth analysis of RBPs and other related players will unveil the sophisticated regulatory mechanisms of RNA processing during plant immune responses.

    4. The Old and New RNA World

      Directory of Open Access Journals (Sweden)

      Zofia Szweykowska-Kulińska

      2014-12-01

      Full Text Available Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we

    5. Small catalytic RNA: Structure, function and application

      Energy Technology Data Exchange (ETDEWEB)

      Monforte, J.A.

      1991-04-01

      We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

    6. Inverse folding of RNA pseudoknot structures

      Directory of Open Access Journals (Sweden)

      Li Linda YM

      2010-06-01

      Full Text Available Abstract Background RNA exhibits a variety of structural configurations. Here we consider a structure to be tantamount to the noncrossing Watson-Crick and G-U-base pairings (secondary structure and additional cross-serial base pairs. These interactions are called pseudoknots and are observed across the whole spectrum of RNA functionalities. In the context of studying natural RNA structures, searching for new ribozymes and designing artificial RNA, it is of interest to find RNA sequences folding into a specific structure and to analyze their induced neutral networks. Since the established inverse folding algorithms, RNAinverse, RNA-SSD as well as INFO-RNA are limited to RNA secondary structures, we present in this paper the inverse folding algorithm Inv which can deal with 3-noncrossing, canonical pseudoknot structures. Results In this paper we present the inverse folding algorithm Inv. We give a detailed analysis of Inv, including pseudocodes. We show that Inv allows to design in particular 3-noncrossing nonplanar RNA pseudoknot 3-noncrossing RNA structures-a class which is difficult to construct via dynamic programming routines. Inv is freely available at http://www.combinatorics.cn/cbpc/inv.html. Conclusions The algorithm Inv extends inverse folding capabilities to RNA pseudoknot structures. In comparison with RNAinverse it uses new ideas, for instance by considering sets of competing structures. As a result, Inv is not only able to find novel sequences even for RNA secondary structures, it does so in the context of competing structures that potentially exhibit cross-serial interactions.

    7. Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

      Science.gov (United States)

      Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

      2010-11-01

      MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

    8. RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction

      Science.gov (United States)

      Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

      2014-01-01

      Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA–RNA/RNA–protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA–RNA/RNA–protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA–RNA/RNA–protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

    9. Peptides as catalysts in the RNA world

      DEFF Research Database (Denmark)

      Wieczorek, Rafal; Dörr, Mark; Luisi, Pier Luigi

      The emergence of RNA chains from prebiotic soup is considered a stumbling block in the RNA world theory (Orgel 2004). Both the activation of RNA monomers and their subsequent oligomerization is hard to achieve in accepted early Earth conditions, thus putting doubt on the prebiotic plausibility...... chemistry and the RNA world. Prebiotic soup likely contained complex mixtures of various molecules. Interaction of peptides and nucleotides shows that we should give more consideration to systems chemistry approach in the origin-of-life research. Gorlero M, Wieczorek R, Adamala K, Giorgi A, Schininà ME...

    10. Emerging connections between RNA and autophagy

      DEFF Research Database (Denmark)

      Frankel, Lisa B; Lubas, Michal; Lund, Anders H

      2017-01-01

      in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge......Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority...... of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease....

    11. MiRNA Biogenesis and Intersecting Pathways

      DEFF Research Database (Denmark)

      Ben Chaabane, Samir

      MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Plant miRNAs are critical for plant growth, development and stress response, and are processed in Arabidopsis from primary miRNA transcripts (pri-miRNAs) by the endonuclease activity of the DICER-LIKE1...... questions need to be addressed to establish a valid link, we provide encouraging evidence of the involvement of chromatin remodeling factors FAS1 and FAS2 in miRNA biogenesis. Together, we have expanded our understanding of the intersections between miRNA biogenesis and other pathways....

    12. Personalized RNA Medicine for Pancreatic Cancer.

      Science.gov (United States)

      Gilles, Maud-Emmanuelle; Hao, Liangliang; Huang, Ling; Rupaimoole, Rajesha; Lopez-Casas, Pedro P; Pulver, Emilia; Jeong, Jong Cheol; Muthuswamy, Senthil K; Hidalgo, Manuel; Bhatia, Sangeeta N; Slack, Frank J

      2018-04-01

      Purpose: Since drug responses vary between patients, it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. In this study, we tested whether RNA-based therapeutics were suitable for personalized medicine by using patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models. Experimental Design: We performed microRNA (miRNA) profiling of PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients. To deliver personalized RNA-based-therapy targeting oncogenic miRNAs that form part of this common PDAC miRNA over-expression signature, we packaged antimiR oligonucleotides against one of these miRNAs in tumor-penetrating nanocomplexes (TPN) targeting cell surface proteins on PDAC tumors. Results: As a validation for our pre-clinical strategy, the therapeutic potential of one of our nano-drugs, TPN-21, was first shown to decrease tumor cell growth and survival in PDO avatars for individual patients, then in their PDX avatars. Conclusions: This general approach appears suitable for co-clinical validation of personalized RNA medicine and paves the way to prospectively identify patients with eligible miRNA profiles for personalized RNA-based therapy. Clin Cancer Res; 24(7); 1734-47. ©2018 AACR . ©2018 American Association for Cancer Research.

    13. mRNA processing in yeast

      International Nuclear Information System (INIS)

      Stevens, A.

      1982-01-01

      Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

    14. MicroRNA delivery for regenerative medicine.

      Science.gov (United States)

      Peng, Bo; Chen, Yongming; Leong, Kam W

      2015-07-01

      MicroRNA (miRNA) directs post-transcriptional regulation of a network of genes by targeting mRNA. Although relatively recent in development, many miRNAs direct differentiation of various stem cells including induced pluripotent stem cells (iPSCs), a major player in regenerative medicine. An effective and safe delivery of miRNA holds the key to translating miRNA technologies. Both viral and nonviral delivery systems have seen success in miRNA delivery, and each approach possesses advantages and disadvantages. A number of studies have demonstrated success in augmenting osteogenesis, improving cardiogenesis, and reducing fibrosis among many other tissue engineering applications. A scaffold-based approach with the possibility of local and sustained delivery of miRNA is particularly attractive since the physical cues provided by the scaffold may synergize with the biochemical cues induced by miRNA therapy. Herein, we first briefly cover the application of miRNA to direct stem cell fate via replacement and inhibition therapies, followed by the discussion of the promising viral and nonviral delivery systems. Next we present the unique advantages of a scaffold-based delivery in achieving lineage-specific differentiation and tissue development. Copyright © 2015 Elsevier B.V. All rights reserved.

    15. Messenger RNA 3' end formation in plants.

      Science.gov (United States)

      Hunt, A G

      2008-01-01

      Messenger RNA 3' end formation is an integral step in the process that gives rise to mature, translated messenger RNAs in eukaryotes. With this step, a pre-messenger RNA is processed and polyadenylated, giving rise to a mature mRNA bearing the characteristic poly(A) tract. The poly(A) tract is a fundamental feature of mRNAs, participating in the process of translation initiation and being the focus of control mechanisms that define the lifetime of mRNAs. Thus messenger RNA 3' end formation impacts two steps in mRNA biogenesis and function. Moreover, mRNA 3' end formation is something of a bridge that integrates numerous other steps in mRNA biogenesis and function. While the process is essential for the expression of most genes, it is also one that is subject to various forms of regulation, such that both quantitative and qualitative aspects of gene expression may be modulated via the polyadenylation complex. In this review, the current status of understanding of mRNA 3' end formation in plants is discussed. In particular, the nature of mRNA 3' ends in plants is reviewed, as are recent studies that are beginning to yield insight into the functioning and regulation of plant polyadenylation factor subunits.

    16. Exploring RNA structure by integrative molecular modelling

      DEFF Research Database (Denmark)

      Masquida, Benoît; Beckert, Bertrand; Jossinet, Fabrice

      2010-01-01

      RNA molecular modelling is adequate to rapidly tackle the structure of RNA molecules. With new structured RNAs constituting a central class of cellular regulators discovered every year, the need for swift and reliable modelling methods is more crucial than ever. The pragmatic method based...... on interactive all-atom molecular modelling relies on the observation that specific structural motifs are recurrently found in RNA sequences. Once identified by a combination of comparative sequence analysis and biochemical data, the motifs composing the secondary structure of a given RNA can be extruded...

    17. The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

      Science.gov (United States)

      Ambardekar, Vishakha V; Han, Huai-Yun; Varney, Michelle L; Vinogradov, Serguei V; Singh, Rakesh K; Vetro, Joseph A

      2011-02-01

      Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. Copyright © 2010 Elsevier Ltd. All rights reserved.

    18. The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

      Science.gov (United States)

      Ambardekar, Vishakha V.; Han, Huai-Yun; Varney, Michelle L.; Vinogradov, Serguei V.; Singh, Rakesh K.; Vetro, Joseph A.

      2010-01-01

      Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3′ cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. PMID:21047680

    19. Effect of the nucleotides surrounding the start codon on the translation of foot-and-mouth disease virus RNA.

      Science.gov (United States)

      Ma, X X; Feng, Y P; Gu, Y X; Zhou, J H; Ma, Z R

      2016-06-01

      As for the alternative AUGs in foot-and-mouth disease virus (FMDV), nucleotide bias of the context flanking the AUG(2nd) could be used as a strong signal to initiate translation. To determine the role of the specific nucleotide context, dicistronic reporter constructs were engineered to contain different versions of nucleotide context linking between internal ribosome entry site (IRES) and downstream gene. The results indicate that under FMDV IRES-dependent mechanism, the nucleotide contexts flanking start codon can influence the translation initiation efficiencies. The most optimal sequences for both start codons have proved to be UUU AUG(1st) AAC and AAG AUG(2nd) GAA.

    20. RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

      Directory of Open Access Journals (Sweden)

      Isabel X. Wang

      2014-03-01

      Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

    1. RNA three-way junctions can act as flexible RNA structural elements in large RNA molecules: a molecular simulation analysis

      Czech Academy of Sciences Publication Activity Database

      Beššeová, Ivana; Réblová, Kamila; Leontis, N.B.; Šponer, Jiří

      2009-01-01

      Roč. 26, č. 6 (2009), s. 830-831 ISSN 0739-1102. [The 17th Conversation . 16.06.2009-20.06.2009, Albany] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : RNA three-way junctions * RNA Subject RIV: BO - Biophysics

    2. The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation

      NARCIS (Netherlands)

      Abbink, Truus E. M.; Ooms, Marcel; Haasnoot, P. C. Joost; Berkhout, Ben

      2005-01-01

      The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a

    3. In vitro transcription of Sonchus yellow net virus RNA by a virus-associated RNA-dependent RNA polymerase

      NARCIS (Netherlands)

      Flore, P.H.

      1986-01-01

      The aim of the investigation presented in this thesis was to elucidate the nature of the RNA- dependent RNA polymerase, thought to be associated with Sonchus yellow net virus (SYNV), a rhabdovirus infecting plants. This research was initiated to shed light on the

    4. [Satellite RNA (RNA3) of tomato black ring virus is found with one of the 2 major RNAs (RNA2) in a new capsid nucleoprotein].

      Science.gov (United States)

      Doz, B; Dunez, J; Bove, J M

      1977-12-19

      Tomato Black Ring Virus (TBRV) like other NEPOviruses posseses two nucleoproteins M and B and two major RNAs, RNA1 and RNA2 respectively distributed in B and M. A new nucleoprotein has just been discovered and comprises one molecule of RNA2 associated with one molecule of RNA3. RNA3 is a small RNA of molecular weight 500,000 d considered to be a satellite RNA. Its level appears to depend on the infection stage, local or systemic. RNA3 is able to modify the relative proportions of nucleoproteins M and B and their respective RNAs. The satellite RNA, might be part of the genome and represent a monocistronic mRNA for protein capsid synthesis. However it seems perhaps more tempting to correlate TBRV-RNA3 with satellite RNA5 of certain strains of Cucumber mosaic virus.

    5. Alterations in messenger RNA and small nuclear RNA metabolism resulting from fluorouracil incorporation

      International Nuclear Information System (INIS)

      Armstrong, R.D.; Cadman, E.C.

      1985-01-01

      Studies were completed to examine the effect of 5-fluorouracil (FUra) incorporation on messenger RNA (mRNA) and small molecular weight nuclear RNA (SnRNA) metabolism. Studies of mRNA were completed using cDNA-mRNA hybridization methods to specifically examine dihydrofolate reductase (DHFR) mRNA. C 3 -L5178Y murine leukemia cells which are gene-amplified for DHFR, were exposed to FUra for 6, 12 or 24 hr, and the nuclear and cytoplasmic levels of DHFR-mRNA determined by hybridization with 32 P-DHFR-cDNA. FUra produced a dose-dependent increase in nuclear DHFR-mRNA levels, while total cytoplasmic DHFR-mRNA levels appeared to be unchanged. To examine only mRNA synthesized during FUra exposure, cells were also treated concurrently with [ 3 H] cytidine, and the [ 3 H]mRNA-cDNA hybrids measured following S 1 -nuclease treatment. FUra produced a concentration-dependent increase in nascent nuclear DHFR-mRNA levels, and a decrease in nascent cytoplasmic DHFR-mRNAs levels. These results suggest that FUra produces either an inhibition of mRNA processing, or an inhibition of nuclear-cytoplasmic transport. Preliminary experiments to examine ATP-dependent mRNA transport were completed with isolated nuclei from cells treated with FUra for 1 or 24 hr and then pulse-labeled for 1 hr with [ 3 H] cytidine. The results demonstrate a FUra-concentration and time-dependent inhibition of ATP-mediated mRNA efflux

    6. Mapping protein-RNA interactions by RCAP, RNA-cross-linking and peptide fingerprinting.

      Science.gov (United States)

      Vaughan, Robert C; Kao, C Cheng

      2015-01-01

      RNA nanotechnology often feature protein RNA complexes. The interaction between proteins and large RNAs are difficult to study using traditional structure-based methods like NMR or X-ray crystallography. RCAP, an approach that uses reversible-cross-linking affinity purification method coupled with mass spectrometry, has been developed to map regions within proteins that contact RNA. This chapter details how RCAP is applied to map protein-RNA contacts within virions.

    7. Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles.

      Science.gov (United States)

      Haque, Farzin; Guo, Peixuan

      2015-01-01

      RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149-155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833-842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applications in nanomedicine and nanotechnology. Rapid advances in RNA chemistry, RNA biophysics, and RNA biology have created new opportunities for translating basic science into clinical practice. RNA nanotechnology holds considerable promise in this regard. Increased evidence also suggests that substantial part of the 98.5 % of human genome (Lander et al. Nature 409:860-921, 2001) that used to be called "junk DNA" actually codes for noncoding RNA. As we understand more on how RNA structures are related to function, we can fabricate synthetic RNA nanoparticles for the diagnosis and treatment of diseases. This chapter provides a brief overview of the field regarding the design, construction, purification, and characterization of RNA nanoparticles for diverse applications in nanotechnology and nanomedicince.

    8. Methylated nucleosides in tRNA and tRNA methyltransferases

      Directory of Open Access Journals (Sweden)

      Hiroyuki eHori

      2014-05-01

      Full Text Available To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon–anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.

    9. Mutant allele of rna14 in fission yeast affects pre-mRNA splicing

      Indian Academy of Sciences (India)

      transcript. Rna14 protein in budding yeast has been implicated in cleavage and ... Subsequently, genetic interaction of Rna14 with prp1 and physical .... molecular yeast techniques as described by Moreno et al. ..... To elucidate the role of Rna14 in splicing, RT-PCR analysis ..... design principles of a dynamic RNP machine.

    10. Signatures of RNA binding proteins globally coupled to effective microRNA target sites

      DEFF Research Database (Denmark)

      Jacobsen, Anders; Wen, Jiayu; Marks, Debora S

      2010-01-01

      MicroRNAs (miRNAs) and small interfering RNAs (siRNAs), bound to Argonaute proteins (RISC), destabilize mRNAs through base-pairing with the mRNA. However, the gene expression changes after perturbations of these small RNAs are only partially explained by predicted miRNA/siRNA targeting. Targeting...

    11. RNA trafficking in parasitic plant systems

      Science.gov (United States)

      LeBlanc, Megan; Kim, Gunjune; Westwood, James H.

      2012-01-01

      RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host–parasite connections and the potential significance of host RNAs for the parasite. Additional research on host–parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking. PMID:22936942

    12. RNA Encapsidation and Packaging in the Phleboviruses

      Directory of Open Access Journals (Sweden)

      Katherine E. Hornak

      2016-07-01

      Full Text Available The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV, severe fever with thrombocytopenia syndrome virus (SFTSV, Uukuniemi virus (UUKV, and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5–7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA are synthesized. The interaction between the vRNA and the viral nucleocapsid (N protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses.

    13. RNA trafficking in parasitic plant systems

      Directory of Open Access Journals (Sweden)

      Megan L LeBlanc

      2012-08-01

      Full Text Available RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host parasite connections and the potential significance of host RNAs for the parasite. Additional research on host-parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking.

    14. Customization of Artificial MicroRNA Design.

      Science.gov (United States)

      Van Vu, Tien; Do, Vinh Nang

      2017-01-01

      RNAi approaches, including microRNA (miRNA) regulatory pathway, offer great tools for functional characterization of unknown genes. Moreover, the applications of artificial microRNA (amiRNA) in the field of plant transgenesis have also been advanced to engineer pathogen-resistant or trait-improved transgenic plants. Until now, despite the high potency of amiRNA approach, no commercial plant cultivar expressing amiRNAs with improved traits has been released yet. Beside the issues of biosafety policies, the specificity and efficacy of amiRNAs are of major concerns. Sufficient cares should be taken for the specificity and efficacy of amiRNAs due to their potential off-target effects and other issues relating to in vivo expression of pre-amiRNAs. For these reasons, the proper design of amiRNAs with the lowest off-target possibility is very important for successful applications of the approach in plant. Therefore, there are many studies with the aim to improve the amiRNA design and amiRNA expressing backbones for obtaining better specificity and efficacy. However, the requirement for an efficient reference for the design is still needed. In the present chapter, we attempt to summarize and discuss all the major concerns relating to amiRNA design with the hope to provide a significant guideline for this approach.

    15. Messenger RNA surveillance: neutralizing natural nonsense

      DEFF Research Database (Denmark)

      Weischelfeldt, Joachim Lütken; Lykke-Andersen, Jens; Porse, Bo

      2005-01-01

      Messenger RNA transcripts that contain premature stop codons are degraded by a process termed nonsense-mediated mRNA decay (NMD). Although previously thought of as a pathway that rids the cell of non-functional mRNAs arising from mutations and processing errors, new research suggests a more general...

    16. RNA-based therapies for genodermatoses

      NARCIS (Netherlands)

      Bornert, Olivier; Peking, Patricia; Bremer, Jeroen; Koller, Ulrich; van den Akker, Peter C.; Aartsma-Rus, Annemieke; Pasmooij, Anna M. G.; Murauer, Eva M.; Nystroem, Alexander

      Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA-based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA

    17. ADAR RNA editing below the backbone.

      Science.gov (United States)

      Keegan, Liam; Khan, Anzer; Vukic, Dragana; O'Connell, Mary

      2017-09-01

      ADAR RNA editing enzymes ( a denosine d e a minases acting on R NA) that convert adenosine bases to inosines were first identified biochemically 30 years ago. Since then, studies on ADARs in genetic model organisms, and evolutionary comparisons between them, continue to reveal a surprising range of pleiotropic biological effects of ADARs. This review focuses on Drosophila melanogaster , which has a single Adar gene encoding a homolog of vertebrate ADAR2 that site-specifically edits hundreds of transcripts to change individual codons in ion channel subunits and membrane and cytoskeletal proteins. Drosophila ADAR is involved in the control of neuronal excitability and neurodegeneration and, intriguingly, in the control of neuronal plasticity and sleep. Drosophila ADAR also interacts strongly with RNA interference, a key antiviral defense mechanism in invertebrates. Recent crystal structures of human ADAR2 deaminase domain-RNA complexes help to interpret available information on Drosophila ADAR isoforms and on the evolution of ADARs from tRNA deaminase ADAT proteins. ADAR RNA editing is a paradigm for the now rapidly expanding range of RNA modifications in mRNAs and ncRNAs. Even with recent progress, much remains to be understood about these groundbreaking ADAR RNA modification systems. © 2017 Keegan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

    18. Retroviral RNA Dimerization: From Structure to Functions

      Directory of Open Access Journals (Sweden)

      Noé Dubois

      2018-03-01

      Full Text Available The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…, the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.

    19. The integrated analysis of RNA-seq and microRNA-seq depicts miRNA-mRNA networks involved in Japanese flounder (Paralichthys olivaceus) albinism.

      Science.gov (United States)

      Wang, Na; Wang, Ruoqing; Wang, Renkai; Tian, Yongsheng; Shao, Changwei; Jia, Xiaodong; Chen, Songlin

      2017-01-01

      Albinism, a phenomenon characterized by pigmentation deficiency on the ocular side of Japanese flounder (Paralichthys olivaceus), has caused significant damage. Limited mRNA and microRNA (miRNA) information is available on fish pigmentation deficiency. In this study, a high-throughput sequencing strategy was employed to identify the mRNA and miRNAs involved in P. olivaceus albinism. Based on P. olivaceus genome, RNA-seq identified 21,787 know genes and 711 new genes by transcripts assembly. Of those, 235 genes exhibited significantly different expression pattern (fold change ≥2 or ≤0.5 and q-value≤0.05), including 194 down-regulated genes and 41 up-regulated genes in albino versus normally pigmented individuals. These genes were enriched to 81 GO terms and 9 KEGG pathways (p≤0.05). Among those, the pigmentation related pathways-Melanogenesis and tyrosine metabolism were contained. High-throughput miRNA sequencing identified a total of 475 miRNAs, including 64 novel miRNAs. Furthermore, 33 differentially expressed miRNAs containing 13 up-regulated and 20 down-regulated miRNAs were identified in albino versus normally pigmented individuals (fold change ≥1.5 or ≤0.67 and p≤0.05). The next target prediction discovered a variety of putative target genes, of which, 134 genes including Tyrosinase (TYR), Tyrosinase-related protein 1 (TYRP1), Microphthalmia-associated transcription factor (MITF) were overlapped with differentially expressed genes derived from RNA-seq. These target genes were significantly enriched to 254 GO terms and 103 KEGG pathways (p<0.001). Of those, tyrosine metabolism, lysosomes, phototransduction pathways, etc., attracted considerable attention due to their involvement in regulating skin pigmentation. Expression patterns of differentially expressed mRNA and miRNAs were validated in 10 mRNA and 10 miRNAs by qRT-PCR. With high-throughput mRNA and miRNA sequencing and analysis, a series of interested mRNA and miRNAs involved in fish

    20. Long noncoding RNA in hematopoiesis and immunity.

      Science.gov (United States)

      Satpathy, Ansuman T; Chang, Howard Y

      2015-05-19

      Dynamic gene expression during cellular differentiation is tightly coordinated by transcriptional and post-transcriptional mechanisms. An emerging theme is the central role of long noncoding RNAs (lncRNAs) in the regulation of this specificity. Recent advances demonstrate that lncRNAs are expressed in a lineage-specific manner and control the development of several cell types in the hematopoietic system. Moreover, specific lncRNAs are induced to modulate innate and adaptive immune responses. lncRNAs can function via RNA-DNA, RNA-RNA, and RNA-protein target interactions. As a result, they affect several stages of gene regulation, including chromatin modification, mRNA biogenesis, and protein signaling. We discuss recent advances, future prospects, and challenges in understanding the roles of lncRNAs in immunity and immune-mediated diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

    1. A probabilistic model of RNA conformational space

      DEFF Research Database (Denmark)

      Frellsen, Jes; Moltke, Ida; Thiim, Martin

      2009-01-01

      , the discrete nature of the fragments necessitates the use of carefully tuned, unphysical energy functions, and their non-probabilistic nature impairs unbiased sampling. We offer a solution to the sampling problem that removes these important limitations: a probabilistic model of RNA structure that allows...... conformations for 9 out of 10 test structures, solely using coarse-grained base-pairing information. In conclusion, the method provides a theoretical and practical solution for a major bottleneck on the way to routine prediction and simulation of RNA structure and dynamics in atomic detail.......The increasing importance of non-coding RNA in biology and medicine has led to a growing interest in the problem of RNA 3-D structure prediction. As is the case for proteins, RNA 3-D structure prediction methods require two key ingredients: an accurate energy function and a conformational sampling...

    2. Eukaryotic 5S rRNA biogenesis

      Science.gov (United States)

      Ciganda, Martin; Williams, Noreen

      2012-01-01

      The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. PMID:21957041

    3. RNA as a small molecule druggable target.

      Science.gov (United States)

      Rizvi, Noreen F; Smith, Graham F

      2017-12-01

      Small molecule drugs have readily been developed against many proteins in the human proteome, but RNA has remained an elusive target for drug discovery. Increasingly, we see that RNA, and to a lesser extent DNA elements, show a persistent tertiary structure responsible for many diverse and complex cellular functions. In this digest, we have summarized recent advances in screening approaches for RNA targets and outlined the discovery of novel, drug-like small molecules against RNA targets from various classes and therapeutic areas. The link of structure, function, and small-molecule Druggability validates now for the first time that RNA can be the targets of therapeutic agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

    4. MicroRNA Implication in Cancer

      Directory of Open Access Journals (Sweden)

      Iker BADIOLA

      2010-03-01

      Full Text Available MicroRNAs (miRNA are a new class of posttranscriptional regulators. These small non-coding RNAs regulate the expression of target mRNA transcripts and are linked to several human disease such as Alzheimer, cancer or heart disease. But it has been the cancer disease which has experimented the major number of studies of miRNA linked to the disease progression. In the last years it has been reported the deregulation pattern of the miRNAs in malignant cells which have disrupted the control of the proliferation, differentiation or apoptosis. The evidence of the presence of specific miRNA deregulated in concrete cancer types has become the miRNAs like possible biomarkers and therapeutic targets. The specific miRNA patterns deregulated in concrete cancer cell types open new opportunities to the diagnosis and therapy.

    5. Transfer RNA methylases in rat placenta

      International Nuclear Information System (INIS)

      Jagtiani, S.K.; Narurkar, L.M.; Narurkar, M.V.

      1977-01-01

      Presence of tRNA methylases (5-adenosylmethionine : tRNA methyltransferases) was demonstrated at various stages of gestation in rat placenta, the enzyme being 50-100% higher than that of adult rat liver during early gestation. Placental tRNA methylases were shown to differ from those of liver in the extent of methylation. Glycine methyltransferase (S-adenosylmethionine : glycine methyltransferase), a regulatory enzyme in adult rat liver, was absent in placenta throughout gestation. The placental tRNA methylases could be inhibited in vitro by semipurified glycine methyltransferase from adult rat liver. The high placental tRNA methylase activity was comparable with the inhibitor-free enzyme activity of the adult rat liver. S-adenosyl-[Me- 14 C]-methionine was used in the investigation. (author)

    6. Identifying and characterizing Hfq-RNA interactions.

      Science.gov (United States)

      Faner, M A; Feig, A L

      2013-09-15

      To regulate stress responses and virulence, bacteria use small regulatory RNAs (sRNAs). These RNAs can up or down regulate target mRNAs through base pairing by influencing ribosomal access and RNA decay. A large class of these sRNAs, called trans-encoded sRNAs, requires the RNA binding protein Hfq to facilitate base pairing between the regulatory RNA and its target mRNA. The resulting network of regulation is best characterized in Escherichia coli and Salmonella typhimurium, but the importance of Hfq dependent sRNA regulation is recognized in a diverse population of bacteria. In this review we present the approaches and methods used to discover Hfq binding RNAs, characterize their interactions and elucidate their functions. Copyright © 2013 Elsevier Inc. All rights reserved.

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

      Directory of Open Access Journals (Sweden)

      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.

    8. Four RNA families with functional transient structures.

      Science.gov (United States)

      Zhu, Jing Yun A; Meyer, Irmtraud M

      2015-01-01

      Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All

    9. HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication.

      Directory of Open Access Journals (Sweden)

      Jing-Yi Lin

      Full Text Available EV71 (enterovirus 71 RNA contains an internal ribosomal entry site (IRES that directs cap-independent initiation of translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs. We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2 as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication.

    10. RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection

      International Nuclear Information System (INIS)

      Kleinschmidt, A.M.; Pederson, T.

      1990-01-01

      The authors present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32 P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells

    11. MicroRNA-target binding structures mimic microRNA duplex structures in humans.

      Directory of Open Access Journals (Sweden)

      Xi Chen

      Full Text Available Traditionally, researchers match a microRNA guide strand to mRNA sequences using sequence comparisons to predict its potential target genes. However, many of the predictions can be false positives due to limitations in sequence comparison alone. In this work, we consider the association of two related RNA structures that share a common guide strand: the microRNA duplex and the microRNA-target binding structure. We have analyzed thousands of such structure pairs and found many of them share high structural similarity. Therefore, we conclude that when predicting microRNA target genes, considering just the microRNA guide strand matches to gene sequences may not be sufficient--the microRNA duplex structure formed by the guide strand and its companion passenger strand must also be considered. We have developed software to translate RNA binding structure into encoded representations, and we have also created novel automatic comparison methods utilizing such encoded representations to determine RNA structure similarity. Our software and methods can be utilized in the other RNA secondary structure comparisons as well.

    12. Conformational Selection and Induced Fit for RNA Polymerase and RNA/DNA Hybrid Backtracked Recognition

      Directory of Open Access Journals (Sweden)

      Haifeng eChen

      2015-11-01

      Full Text Available RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15 and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS P test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein.

    13. 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.

    14. Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

      Science.gov (United States)

      Hadd, Andrew; Perona, John J

      2014-12-19

      We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

    15. Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication.

      Science.gov (United States)

      Mizumoto, Hiroyuki; Iwakawa, Hiro-Oki; Kaido, Masanori; Mise, Kazuyuki; Okuno, Tetsuro

      2006-04-01

      The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

    16. Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

      Directory of Open Access Journals (Sweden)

      Nicole Ludwig

      2016-03-01

      Full Text Available Wilms tumor (WT is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT.

    17. MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

      Science.gov (United States)

      Corrêa, Régis L; Steiner, Florian A; Berezikov, Eugene; Ketting, René F

      2010-04-08

      RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

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

      Directory of Open Access Journals (Sweden)

      Daniela Toro-Ascuy

      2016-11-01

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

    19. Intranasal delivery of antiviral siRNA.

      Science.gov (United States)

      Barik, Sailen

      2011-01-01

      Intranasal administration of synthetic siRNA is an effective modality of RNAi delivery for the prevention and therapy of respiratory diseases, including pulmonary infections. Vehicles used for nasal siRNA delivery include established as well as novel reagents, many of which have been recently optimized. In general, they all promote significant uptake of siRNA into the lower respiratory tract, including the lung. When properly designed and optimized, these siRNAs offer significant protection against respiratory viruses such as influenza virus, parainfluenza virus and respiratory syncytial virus (RSV). Nasally administered siRNA remains within the lung and does not access systemic blood flow, as judged by its absence in other major organs such as liver, heart, kidney, and skeletal muscle. Adverse immune reaction is generally not encountered, especially when immunogenic and/or off-target siRNA sequences and toxic vehicles are avoided. In fact, siRNA against RSV has entered Phase II clinical trials in human with promising results. Here, we provide a standardized procedure for using the nose as a specific route for siRNA delivery into the lung of laboratory animals. It should be clear that this simple and efficient system has enormous potential for therapeutics.

    20. The cellular receptors of exogenous RNA

      Directory of Open Access Journals (Sweden)

      Patryk Reniewicz

      2016-04-01

      Full Text Available One of the key determinants of survival for organisms is proper recognition of exogenous and endogenous nucleic acids. Therefore, high eukaryotes developed a number of receptors that allow for discrimination between friend or foe DNA and RNA. Appearance of exogenous RNA in cytoplasm provides a signal of danger and triggers cellular responses that facilitate eradication of a pathogen. Recognition of exogenous RNA is additionally complicated by fact that large amount of endogenous RNA is present in cytoplasm Thus, number of different receptors, found in eukaryotic cells, is able to recognize that nucleic acid. First group of those receptors consist endosomal Toll like receptors, namely TLR3, TLR7, TLR8 and TLR13. Those receptors recognize RNA released from pathogens that enter the cell by endocytosis. The second group includes cytoplasmic sensors like PKR and the family of RLRs comprised of RIG-I, MDA5 and LGP2. Cytoplasmic receptors recognize RNA from pathogens invading the cell by non-endocytic pathway. In both cases binding of RNA by its receptors results in activation of the signalling cascades that lead to the production of interferon and other cytokines.

    1. Annotating RNA motifs in sequences and alignments.

      Science.gov (United States)

      Gardner, Paul P; Eldai, Hisham

      2015-01-01

      RNA performs a diverse array of important functions across all cellular life. These functions include important roles in translation, building translational machinery and maturing messenger RNA. More recent discoveries include the miRNAs and bacterial sRNAs that regulate gene expression, the thermosensors, riboswitches and other cis-regulatory elements that help prokaryotes sense their environment and eukaryotic piRNAs that suppress transposition. However, there can be a long period between the initial discovery of a RNA and determining its function. We present a bioinformatic approach to characterize RNA motifs, which are critical components of many RNA structure-function relationships. These motifs can, in some instances, provide researchers with functional hypotheses for uncharacterized RNAs. Moreover, we introduce a new profile-based database of RNA motifs--RMfam--and illustrate some applications for investigating the evolution and functional characterization of RNA. All the data and scripts associated with this work are available from: https://github.com/ppgardne/RMfam. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

    2. RNA interference and Register Machines (extended abstract

      Directory of Open Access Journals (Sweden)

      Masahiro Hamano

      2012-11-01

      Full Text Available RNA interference (RNAi is a mechanism whereby small RNAs (siRNAs directly control gene expression without assistance from proteins. This mechanism consists of interactions between RNAs and small RNAs both of which may be single or double stranded. The target of the mechanism is mRNA to be degraded or aberrated, while the initiator is double stranded RNA (dsRNA to be cleaved into siRNAs. Observing the digital nature of RNAi, we represent RNAi as a Minsky register machine such that (i The two registers hold single and double stranded RNAs respectively, and (ii Machine's instructions are interpreted by interactions of enzyme (Dicer, siRNA (with RISC com- plex and polymerization (RdRp to the appropriate registers. Interpreting RNAi as a computational structure, we can investigate the computational meaning of RNAi, especially its complexity. Initially, the machine is configured as a Chemical Ground Form (CGF, which generates incorrect jumps. To remedy this problem, the system is remodeled as recursive RNAi, in which siRNA targets not only mRNA but also the machine instructional analogues of Dicer and RISC. Finally, probabilistic termination is investigated in the recursive RNAi system.

    3. SRD: a Staphylococcus regulatory RNA database.

      Science.gov (United States)

      Sassi, Mohamed; Augagneur, Yoann; Mauro, Tony; Ivain, Lorraine; Chabelskaya, Svetlana; Hallier, Marc; Sallou, Olivier; Felden, Brice

      2015-05-01

      An overflow of regulatory RNAs (sRNAs) was identified in a wide range of bacteria. We designed and implemented a new resource for the hundreds of sRNAs identified in Staphylococci, with primary focus on the human pathogen Staphylococcus aureus. The "Staphylococcal Regulatory RNA Database" (SRD, http://srd.genouest.org/) compiled all published data in a single interface including genetic locations, sequences and other features. SRD proposes novel and simplified identifiers for Staphylococcal regulatory RNAs (srn) based on the sRNA's genetic location in S. aureus strain N315 which served as a reference. From a set of 894 sequences and after an in-depth cleaning, SRD provides a list of 575 srn exempt of redundant sequences. For each sRNA, their experimental support(s) is provided, allowing the user to individually assess their validity and significance. RNA-seq analysis performed on strains N315, NCTC8325, and Newman allowed us to provide further details, upgrade the initial annotation, and identified 159 RNA-seq independent transcribed sRNAs. The lists of 575 and 159 sRNAs sequences were used to predict the number and location of srns in 18 S. aureus strains and 10 other Staphylococci. A comparison of the srn contents within 32 Staphylococcal genomes revealed a poor conservation between species. In addition, sRNA structure predictions obtained with MFold are accessible. A BLAST server and the intaRNA program, which is dedicated to target prediction, were implemented. SRD is the first sRNA database centered on a genus; it is a user-friendly and scalable device with the possibility to submit new sequences that should spread in the literature. © 2015 Sassi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

    4. Circular RNA (circRNA) was an important bridge in the switch from the RNA world to the DNA world.

      Science.gov (United States)

      Soslau, Gerald

      2018-06-14

      The concept that life on Earth began as an RNA world has been built upon extensive experimentation demonstrating that many of the building blocks required for living cells could be synthesized in the laboratory under conditions approximating our primordial world. Many of the building blocks for life have also been found in meteorites indicating that meteors may have been a source for these molecules, or more likely, that they represent the chemical library present in most/all bodies in the universe after the big bang. Perhaps the most important support for the concept comes from the fact that some RNA species possess catalytic activity, ribozymes, and that RNA could be reverse transcribe to DNA. The thrust of numerous papers on this topic has been to explore how the available molecules on Earth, at its birth, gave rise to life as we know it today. This paper focuses more on a reverse view of the topic. The "how" molecular building blocks were synthesized is not addressed nor how the "first" RNA molecules were synthesized. We can clearly speculate on the variable environmental conditions and chemistry available on Earth billions of years ago. However, we can never truly replicate the changing conditions or know the chemical composition of Earth at the beginning of time. We can, however, confirm that over millions, perhaps billions of years the basic building blocks for life accumulated sufficiently to initiate evolution to an RNA world followed by our RNA/DNA world. Here we are attempting to take the information from our current knowledge of biology and by inference and extrapolation work backward to hypothesize biological events in the march forward from RNA to DNA. It is proposed that the primordial replicating RNA cell, the ribocyte, evolved from liposomes encompassing required reactants and products for "life" and that ribonucleopeptide complexes formed membrane pores to support bidirectional ion and molecular transport to maintain biological functions and

    5. Chemical fidelity of an RNA polymerase ribozyme

      DEFF Research Database (Denmark)

      Attwater, J.; Tagami, S.; Kimoto, M.

      2013-01-01

      for function. Here we have explored the chemical fidelity, i.e. substrate selectivity and specificity for both single and multiple catalytic steps of the Z RNA polymerase ribozyme-a modern day analogue of the primordial RNA replicase. Using a wide range of nucleotide analogues and ionic conditions, we observe......The emergence of catalytically active RNA enzymes (ribozymes) is widely believed to have been an important transition in the origin of life. In the context of a likely heterogeneous chemical environment, substrate specificity and selectivity of these primordial enzymes would have been critical...

    6. Exploring complex miRNA-mRNA interactions with Bayesian networks by splitting-averaging strategy

      Directory of Open Access Journals (Sweden)

      Liu Lin

      2009-12-01

      Full Text Available Abstract Background microRNAs (miRNAs regulate target gene expression by controlling their mRNAs post-transcriptionally. Increasing evidence demonstrates that miRNAs play important roles in various biological processes. However, the functions and precise regulatory mechanisms of most miRNAs remain elusive. Current research suggests that miRNA regulatory modules are complicated, including up-, down-, and mix-regulation for different physiological conditions. Previous computational approaches for discovering miRNA-mRNA interactions focus only on down-regulatory modules. In this work, we present a method to capture complex miRNA-mRNA interactions including all regulatory types between miRNAs and mRNAs. Results We present a method to capture complex miRNA-mRNA interactions using Bayesian network structure learning with splitting-averaging strategy. It is designed to explore all possible miRNA-mRNA interactions by integrating miRNA-targeting information, expression profiles of miRNAs and mRNAs, and sample categories. We also present an analysis of data sets for epithelial and mesenchymal transition (EMT. Our results show that the proposed method identified all possible types of miRNA-mRNA interactions from the data. Many interactions are of tremendous biological significance. Some discoveries have been validated by previous research, for example, the miR-200 family negatively regulates ZEB1 and ZEB2 for EMT. Some are consistent with the literature, such as LOX has wide interactions with the miR-200 family members for EMT. Furthermore, many novel interactions are statistically significant and worthy of validation in the near future. Conclusions This paper presents a new method to explore the complex miRNA-mRNA interactions for different physiological conditions using Bayesian network structure learning with splitting-averaging strategy. The method makes use of heterogeneous data including miRNA-targeting information, expression profiles of miRNAs and

    7. Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

      Science.gov (United States)

      Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

      2018-01-01

      Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

    8. Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

      Science.gov (United States)

      Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

      2007-04-01

      During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

    9. Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

      Directory of Open Access Journals (Sweden)

      Alexey Berezhnoy

      2012-01-01

      Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

    10. A Method to Predict the Structure and Stability of RNA/RNA Complexes.

      Science.gov (United States)

      Xu, Xiaojun; Chen, Shi-Jie

      2016-01-01

      RNA/RNA interactions are essential for genomic RNA dimerization and regulation of gene expression. Intermolecular loop-loop base pairing is a widespread and functionally important tertiary structure motif in RNA machinery. However, computational prediction of intermolecular loop-loop base pairing is challenged by the entropy and free energy calculation due to the conformational constraint and the intermolecular interactions. In this chapter, we describe a recently developed statistical mechanics-based method for the prediction of RNA/RNA complex structures and stabilities. The method is based on the virtual bond RNA folding model (Vfold). The main emphasis in the method is placed on the evaluation of the entropy and free energy for the loops, especially tertiary kissing loops. The method also uses recursive partition function calculations and two-step screening algorithm for large, complicated structures of RNA/RNA complexes. As case studies, we use the HIV-1 Mal dimer and the siRNA/HIV-1 mutant (T4) to illustrate the method.

    11. Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

      International Nuclear Information System (INIS)

      Panaviene, Zivile; Nagy, Peter D.

      2003-01-01

      RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination

    12. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

      Science.gov (United States)

      Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

      2016-01-01

      Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

    13. Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

      Science.gov (United States)

      Li, Xin; Ben-Dov, Iddo Z; Mauro, Maurizio; Williams, Zev

      2015-05-06

      RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

    14. siRNA as an alternative therapy against viral infections

      Directory of Open Access Journals (Sweden)

      Hana A. Pawestri

      2012-07-01

      Full Text Available siRNA (small interfering ribonucleic acid adalah sebuah metode yang dapat digunakan untuk mengatasi infeksi virus yang prinsip kerjanya berdasarkan metode komplementer dsRNA (double stranded RNA pada RNA virus sehingga menyebabkan kegagalan proses transkripsi (silencing.  Untuk lebih memahami bagaimana proses kerja dan ulasan penelitian siRNA yang terkini, di dalam tulisan ini ditinjau siRNA sebagai metoda yang dikembangkan untuk mengatasi infeksi dan meneliti efeknya pada replikasi beberapa virus seperti Hepatitis C, Influenza, Polio, dan HIV. Kami menemukan bahwa urutan basa nukleotida dari target siRNA sangat penting. Hal tersebut harus homolog dengan target RNA virus dan tidak menganggu RNA sel inang. Untuk mengurangi kegagalan terapi siRNA oleh adanya mutasi, digunakan beberapa siRNA yang sekaligus menjadi target RNA virus yang berbeda. Namun demikian, terapi siRNA masih menghadapi beberapa kesulitan seperti pengiriman (transfer khusus ke jaringan yang terinfeksi dan perlindungan siRNA dari perusakan oleh nuklease. Berdasarkan beberapa penelitian yang telah dilakukan, siRNA dapat digunakan sebagai alternatif untuk mengobati infeksi yang disebabkan oleh virus. Terapi tersebut direkomendasikan untuk dilakukan uji klinis dengan memperhatikan beberapa aspek seperti desain siRNA dan mekanisme transfer. (Health Science Indones 2010; 1: 58 - 65 Kata kunci: siRNA, infeksi virus, target virus, alternatif terapi Abstract SiRNA is a promising method to deal with viral infections. The principle of siRNA is based on the complementarily of (synthetic dsRNA to an RNA virus which, in consequence, will be silenced. Many studies are currently examining the effects of siRNA on replication of diverse virus types like Hepatitis C, polio and HIV. The choice of the siRNA target sequence is crucial. It has to be very homologous to the target RNA, but it cannot target RNA of the host cell. To reduce the possibility for the virus to escape from the siRNA therapy by

    15. Mapping RNA-seq Reads with STAR.

      Science.gov (United States)

      Dobin, Alexander; Gingeras, Thomas R

      2015-09-03

      Mapping of large sets of high-throughput sequencing reads to a reference genome is one of the foundational steps in RNA-seq data analysis. The STAR software package performs this task with high levels of accuracy and speed. In addition to detecting annotated and novel splice junctions, STAR is capable of discovering more complex RNA sequence arrangements, such as chimeric and circular RNA. STAR can align spliced sequences of any length with moderate error rates, providing scalability for emerging sequencing technologies. STAR generates output files that can be used for many downstream analyses such as transcript/gene expression quantification, differential gene expression, novel isoform reconstruction, and signal visualization. In this unit, we describe computational protocols that produce various output files, use different RNA-seq datatypes, and utilize different mapping strategies. STAR is open source software that can be run on Unix, Linux, or Mac OS X systems. Copyright © 2015 John Wiley & Sons, Inc.

    16. Facilitating RNA structure prediction with microarrays.

      Science.gov (United States)

      Kierzek, Elzbieta; Kierzek, Ryszard; Turner, Douglas H; Catrina, Irina E

      2006-01-17

      Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.

    17. MicroRNA involvement in glioblastoma pathogenesis

      International Nuclear Information System (INIS)

      Novakova, Jana; Slaby, Ondrej; Vyzula, Rostislav; Michalek, Jaroslav

      2009-01-01

      MicroRNAs are endogenously expressed regulatory noncoding RNAs. Altered expression levels of several microRNAs have been observed in glioblastomas. Functions and direct mRNA targets for these microRNAs have been relatively well studied over the last years. According to these data, it is now evident, that impairment of microRNA regulatory network is one of the key mechanisms in glioblastoma pathogenesis. MicroRNA deregulation is involved in processes such as cell proliferation, apoptosis, cell cycle regulation, invasion, glioma stem cell behavior, and angiogenesis. In this review, we summarize the current knowledge of miRNA functions in glioblastoma with an emphasis on its significance in glioblastoma oncogenic signaling and its potential to serve as a disease biomarker and a novel therapeutic target in oncology.

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

      Science.gov (United States)

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

      2013-01-01

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

    19. Reconstruction and analysis of the lncRNA-miRNA-mRNA network based on competitive endogenous RNA reveal functional lncRNAs in rheumatoid arthritis.

      Science.gov (United States)

      Jiang, Hui; Ma, Rong; Zou, Shubiao; Wang, Yongzhong; Li, Zhuqing; Li, Weiping

      2017-06-01

      Rheumatoid arthritis (RA) is an autoimmune disease with an unknown etiology, occurring in approximately 1.0% of general population. More and more studies have suggested that long non-coding RNAs (lncRNAs) could play important roles in various biological processes and be associated with the pathogenesis of different kinds of diseases including RA. Although a large number of lncRNAs have been found, our knowledge of their function and physiological/pathological significance is still in its infancy. In order to reveal functional lncRNAs and identify the key lncRNAs in RA, we reconstructed a global triple network based on the competitive endogenous RNA (ceRNA) theory using the data from National Center for Biotechnology Information Gene Expression Omnibus and our previous paper. Meanwhile, Gene Ontology (GO) and pathway analysis were performed using Cytoscape plug-in BinGO and Database for Annotation, Visualization, and Integration Discovery (DAVID), respectively. We found that the lncRNA-miRNA-mRNA network was composed of 7 lncRNA nodes, 90 mRNA nodes, 24 miRNA nodes, and 301 edges. The functional assay showed that 147 GO terms and 23 pathways were enriched. In addition, three lncRNAs (S5645.1, XR_006437.1, J01878) were highly related to RA, and therefore, were selected as key lncRNAs. This study suggests that specific lncRNAs are associated with the development of RA, and three lncRNAs (S5645.1, XR_006437.1, J01878) could be used as potential diagnostic biomarkers and therapeutic targets.

    20. Self-amplifying mRNA vaccines.

      Science.gov (United States)

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

      2015-01-01

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

    1. Modelling Toehold-Mediated RNA Strand Displacement

      OpenAIRE

      Šulc, Petr; Ouldridge, Thomas E.; Romano, Flavio; Doye, Jonathan P.K.; Louis, Ard A.

      2015-01-01

      We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperat...

    2. Rfam: updates to the RNA families database

      DEFF Research Database (Denmark)

      Gardner, Paul P; Daub, Jennifer; Tate, John G

      2008-01-01

      Rfam is a collection of RNA sequence families, represented by multiple sequence alignments and covariance models (CMs). The primary aim of Rfam is to annotate new members of known RNA families on nucleotide sequences, particularly complete genomes, using sensitive BLAST filters in combination...... to the website, methodologies and data used by Rfam are discussed. Rfam is freely available on the Web at http://rfam.sanger.ac.uk/and http://rfam.janelia.org/....

    3. RNA search engines empower the bacterial intranet

      OpenAIRE

      Dendooven, T; Luisi, Bonaventura Francesco

      2017-01-01

      RNA acts not only as an information bearer in the biogenesis of proteins from genes, but also as a regulator that participates in the control of gene expression. In bacteria, small RNA molecules (sRNAs) play controlling roles in numerous processes and help to orchestrate complex regulatory networks. Such processes include cell growth and development, response to stress and metabolic change, transcription termination, cell-to-cell communication, and the launching of programmes for host invasio...

    4. Prebiotic RNA Synthesis by Montmorillonite Catalysis

      Directory of Open Access Journals (Sweden)

      Sohan Jheeta

      2014-08-01

      Full Text Available This review summarizes our recent findings on the role of mineral salts in prebiotic RNA synthesis, which is catalyzed by montmorillonite clay minerals. The clay minerals not only catalyze the synthesis of RNA but also facilitate homochiral selection. Preliminary data of these findings have been presented at the “Horizontal Gene Transfer and the Last Universal Common Ancestor (LUCA” conference at the Open University, Milton Keynes, UK, 5–6 September 2013. The objective of this meeting was to recognize the significance of RNA in LUCA. We believe that the prebiotic RNA synthesis from its monomers must have been a simple process. As a first step, it may have required activation of the 5'-end of the mononucleotide with a leaving group, e.g., imidazole in our model reaction (Figure 1. Wide ranges of activating groups are produced from HCN under plausible prebiotic Earth conditions. The final step is clay mineral catalysis in the presence of mineral salts to facilitate selective production of functional RNA. Both the clay minerals and mineral salts would have been abundant on early Earth. We have demonstrated that while montmorillonite (pH 7 produced only dimers from its monomers in water, addition of sodium chloride (1 M enhanced the chain length multifold, as detected by HPLC. The effect of monovalent cations on RNA synthesis was of the following order: Li+ > Na+ > K+. A similar effect was observed with the anions, enhancing catalysis in the following order: Cl− > Br− > I−. The montmorillonite-catalyzed RNA synthesis was not affected by hydrophobic or hydrophilic interactions. We thus show that prebiotic synthesis of RNA from its monomers was a simple process requiring only clay minerals and a small amount of salt.

    5. The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism

      Science.gov (United States)

      Bækkedal, Cecilie; Haugen, Peik

      2015-01-01

      The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding. PMID:26327359

    6. The replisome uses mRNA as a primer after colliding with RNA polymerase.

      Science.gov (United States)

      Pomerantz, Richard T; O'Donnell, Mike

      2008-12-11

      Replication forks are impeded by DNA damage and protein-nucleic acid complexes such as transcribing RNA polymerase. For example, head-on collision of the replisome with RNA polymerase results in replication fork arrest. However, co-directional collision of the replisome with RNA polymerase has little or no effect on fork progression. Here we examine co-directional collisions between a replisome and RNA polymerase in vitro. We show that the Escherichia coli replisome uses the RNA transcript as a primer to continue leading-strand synthesis after the collision with RNA polymerase that is displaced from the DNA. This action results in a discontinuity in the leading strand, yet the replisome remains intact and bound to DNA during the entire process. These findings underscore the notable plasticity by which the replisome operates to circumvent obstacles in its path and may explain why the leading strand is synthesized discontinuously in vivo.

    7. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

      DEFF Research Database (Denmark)

      Peng, Zhiyu; Cheng, Yanbing; Tan, Bertrand Chin-Ming

      2012-01-01

      a computational pipeline that carefully controls for false positives while calling RNA editing events from genome and whole-transcriptome data of the same individual. We identified 22,688 RNA editing events in noncoding genes and introns, untranslated regions and coding sequences of protein-coding genes. Most......RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)(+), poly(A)(-) and small RNA samples. We developed...... changes (∼93%) converted A to I(G), consistent with known editing mechanisms based on adenosine deaminase acting on RNA (ADAR). We also found evidence of other types of nucleotide changes; however, these were validated at lower rates. We found 44 editing sites in microRNAs (miRNAs), suggesting a potential...

    8. The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism.

      Science.gov (United States)

      Bækkedal, Cecilie; Haugen, Peik

      2015-01-01

      The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding.

    9. A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure.

      Science.gov (United States)

      Zhang, Jinwei; Landick, Robert

      2016-04-01

      The vectorial (5'-to-3' at varying velocity) synthesis of RNA by cellular RNA polymerases (RNAPs) creates a rugged kinetic landscape, demarcated by frequent, sometimes long-lived, pauses. In addition to myriad gene-regulatory roles, these pauses temporally and spatially program the co-transcriptional, hierarchical folding of biologically active RNAs. Conversely, these RNA structures, which form inside or near the RNA exit channel, interact with the polymerase and adjacent protein factors to influence RNA synthesis by modulating pausing, termination, antitermination, and slippage. Here, we review the evolutionary origin, mechanistic underpinnings, and regulatory consequences of this interplay between RNAP and nascent RNA structure. We categorize and rationalize the extensive linkage between the transcriptional machinery and its product, and provide a framework for future studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

    10. New windows into retroviral RNA structures.

      Science.gov (United States)

      Jayaraman, Dhivya; Kenyon, Julia Claire

      2018-01-25

      The multiple roles of both viral and cellular RNAs have become increasingly apparent in recent years, and techniques to model them have become significantly more powerful, enabling faster and more accurate visualization of RNA structures. Techniques such as SHAPE (selective 2'OH acylation analysed by primer extension) have revolutionized the field, and have been used to examine RNAs belonging to many and diverse retroviruses. Secondary structure probing reagents such as these have been aided by the development of faster methods of analysis either via capillary or next-generation sequencing, allowing the analysis of entire genomes, and of retroviral RNA structures within virions. Techniques to model the three-dimensional structures of these large RNAs have also recently developed. The flexibility of retroviral RNAs, both structural and functional, is clear from the results of these new experimental techniques. Retroviral RNA structures and structural changes control many stages of the lifecycle, and both the RNA structures themselves and their interactions with ligands are potential new drug targets. In addition, our growing understanding of retroviral RNA structures is aiding our knowledge of cellular RNA form and function.

    11. RNA secondary structure prediction using soft computing.

      Science.gov (United States)

      Ray, Shubhra Sankar; Pal, Sankar K

      2013-01-01

      Prediction of RNA structure is invaluable in creating new drugs and understanding genetic diseases. Several deterministic algorithms and soft computing-based techniques have been developed for more than a decade to determine the structure from a known RNA sequence. Soft computing gained importance with the need to get approximate solutions for RNA sequences by considering the issues related with kinetic effects, cotranscriptional folding, and estimation of certain energy parameters. A brief description of some of the soft computing-based techniques, developed for RNA secondary structure prediction, is presented along with their relevance. The basic concepts of RNA and its different structural elements like helix, bulge, hairpin loop, internal loop, and multiloop are described. These are followed by different methodologies, employing genetic algorithms, artificial neural networks, and fuzzy logic. The role of various metaheuristics, like simulated annealing, particle swarm optimization, ant colony optimization, and tabu search is also discussed. A relative comparison among different techniques, in predicting 12 known RNA secondary structures, is presented, as an example. Future challenging issues are then mentioned.

    12. Bleomycin Can Cleave an Oncogenic Noncoding RNA.

      Science.gov (United States)

      Angelbello, Alicia J; Disney, Matthew D

      2018-01-04

      Noncoding RNAs are pervasive in cells and contribute to diseases such as cancer. A question in biomedical research is whether noncoding RNAs are targets of medicines. Bleomycin is a natural product that cleaves DNA; however, it is known to cleave RNA in vitro. Herein, an in-depth analysis of the RNA cleavage preferences of bleomycin A5 is presented. Bleomycin A5 prefers to cleave RNAs with stretches of AU base pairs. Based on these preferences and bioinformatic analysis, the microRNA-10b hairpin precursor was identified as a potential substrate for bleomycin A5. Both in vitro and cellular experiments demonstrated cleavage. Importantly, chemical cleavage by bleomycin A5 in the microRNA-10b hairpin precursors occurred near the Drosha and Dicer enzymatic processing sites and led to destruction of the microRNA. Evidently, oncogenic noncoding RNAs can be considered targets of cancer medicines and might elicit their pharmacological effects by targeting noncoding RNA. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

    13. RNA Relics and Origin of Life

      Directory of Open Access Journals (Sweden)

      Laurent Vial

      2009-07-01

      Full Text Available A number of small RNA sequences, located in different non-coding sequences and highly preserved across the tree of life, have been suggested to be molecular fossils, of ancient (and possibly primordial origin. On the other hand, recent years have revealed the existence of ubiquitous roles for small RNA sequences in modern organisms, in functions ranging from cell regulation to antiviral activity. We propose that a single thread can be followed from the beginning of life in RNA structures selected only for stability reasons through the RNA relics and up to the current coevolution of RNA sequences; such an understanding would shed light both on the history and on the present development of the RNA machinery and interactions. After presenting the evidence (by comparing their sequences that points toward a common thread, we discuss a scenario of genome coevolution (with emphasis on viral infectious processes and finally propose a plan for the reevaluation of the stereochemical theory of the genetic code; we claim that it may still be relevant, and not only for understanding the origin of life, but also for a comprehensive picture of regulation in present-day cells.

    14. RNA Relics and Origin of Life

      Science.gov (United States)

      Demongeot, Jacques; Glade, Nicolas; Moreira, Andrés; Vial, Laurent

      2009-01-01

      A number of small RNA sequences, located in different non-coding sequences and highly preserved across the tree of life, have been suggested to be molecular fossils, of ancient (and possibly primordial) origin. On the other hand, recent years have revealed the existence of ubiquitous roles for small RNA sequences in modern organisms, in functions ranging from cell regulation to antiviral activity. We propose that a single thread can be followed from the beginning of life in RNA structures selected only for stability reasons through the RNA relics and up to the current coevolution of RNA sequences; such an understanding would shed light both on the history and on the present development of the RNA machinery and interactions. After presenting the evidence (by comparing their sequences) that points toward a common thread, we discuss a scenario of genome coevolution (with emphasis on viral infectious processes) and finally propose a plan for the reevaluation of the stereochemical theory of the genetic code; we claim that it may still be relevant, and not only for understanding the origin of life, but also for a comprehensive picture of regulation in present-day cells. PMID:20111682

    15. RNA Export through the NPC in Eukaryotes.

      Science.gov (United States)

      Okamura, Masumi; Inose, Haruko; Masuda, Seiji

      2015-03-20

      In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.

    16. Small Molecule Modifiers of the microRNA and RNA Interference Pathway

      OpenAIRE

      Deiters, Alexander

      2009-01-01

      Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, whic...

    17. Steric restrictions of RISC in RNA interference identified with size-expanded RNA nucleobases.

      Science.gov (United States)

      Hernández, Armando R; Peterson, Larryn W; Kool, Eric T

      2012-08-17

      Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

    18. Characterizing the transcriptome upon depletion of RNA processing factors

      DEFF Research Database (Denmark)

      Herudek, Jan

      , it is not clear how they target and discriminate their RNA substrates. Moreover, many novel RNA species are poorly characterized and their function is not understood. Over the last decade, protein function has been studied using RNA interference. However, this approach does not allow investigation of instant......The human genome is pervasively transcribed and produces an enormous amount of non-coding RNA (ncRNA). Compared to protein-coding transcripts, many classes of ncRNAs are very unstable and rapidly degraded by the RNA decay machinery. The RNA exosome complex is a main RNA ‘degrader’ in the human...... nucleus and is responsible for the proper processing and decay of a wide range of RNA molecules. Notably, the RNA exosome complex associates with a plethora of co-factors and activators that assist in the recognition of specific RNA substrates. Although many exosome partners have been characterized...

    19. RNA-SeQC: RNA-seq metrics for quality control and process optimization.

      Science.gov (United States)

      DeLuca, David S; Levin, Joshua Z; Sivachenko, Andrey; Fennell, Timothy; Nazaire, Marc-Danie; Williams, Chris; Reich, Michael; Winckler, Wendy; Getz, Gad

      2012-06-01

      RNA-seq, the application of next-generation sequencing to RNA, provides transcriptome-wide characterization of cellular activity. Assessment of sequencing performance and library quality is critical to the interpretation of RNA-seq data, yet few tools exist to address this issue. We introduce RNA-SeQC, a program which provides key measures of data quality. These metrics include yield, alignment and duplication rates; GC bias, rRNA content, regions of alignment (exon, intron and intragenic), continuity of coverage, 3'/5' bias and count of detectable transcripts, among others. The software provides multi-sample evaluation of library construction protocols, input materials and other experimental parameters. The modularity of the software enables pipeline integration and the routine monitoring of key measures of data quality such as the number of alignable reads, duplication rates and rRNA contamination. RNA-SeQC allows investigators to make informed decisions about sample inclusion in downstream analysis. In summary, RNA-SeQC provides quality control measures critical to experiment design, process optimization and downstream computational analysis. See www.genepattern.org to run online, or www.broadinstitute.org/rna-seqc/ for a command line tool.

    20. Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

      Science.gov (United States)

      Ehret, Fabian; Zhou, Cun Yu; Alexander, Seth C; Zhang, Dongyang; Devaraj, Neal K

      2018-03-05

      Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

    1. A Broad RNA Virus Survey Reveals Both miRNA Dependence and Functional Sequestration

      DEFF Research Database (Denmark)

      Scheel, Troels K H; Luna, Joseph M; Liniger, Matthias

      2016-01-01

      , critically depended on the interaction of cellular miR-17 and let-7 with the viral 3' UTR. Unlike canonical miRNA interactions, miR-17 and let-7 binding enhanced pestivirus translation and RNA stability. miR-17 sequestration by pestiviruses conferred reduced AGO binding and functional de...... immunoprecipitation (CLIP) of the Argonaute (AGO) proteins to characterize strengths and specificities of miRNA interactions in the context of 15 different RNA virus infections, including several clinically relevant pathogens. Notably, replication of pestiviruses, a major threat to milk and meat industries...

    2. RNA Interference and its therapeutic applications

      Directory of Open Access Journals (Sweden)

      Srinivasa Rao T

      2011-10-01

      Full Text Available RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS; other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans. [Vet. World 2011; 4(5.000: 225-229

    3. Species-independent MicroRNA Gene Discovery

      KAUST Repository

      Kamanu, Timothy K.

      2012-12-01

      MicroRNA (miRNA) are a class of small endogenous non-coding RNA that are mainly negative transcriptional and post-transcriptional regulators in both plants and animals. Recent studies have shown that miRNA are involved in different types of cancer and other incurable diseases such as autism and Alzheimer’s. Functional miRNAs are excised from hairpin-like sequences that are known as miRNA genes. There are about 21,000 known miRNA genes, most of which have been determined using experimental methods. miRNA genes are classified into different groups (miRNA families). This study reports about 19,000 unknown miRNA genes in nine species whereby approximately 15,300 predictions were computationally validated to contain at least one experimentally verified functional miRNA product. The predictions are based on a novel computational strategy which relies on miRNA family groupings and exploits the physics and geometry of miRNA genes to unveil the hidden palindromic signals and symmetries in miRNA gene sequences. Unlike conventional computational miRNA gene discovery methods, the algorithm developed here is species-independent: it allows prediction at higher accuracy and resolution from arbitrary RNA/DNA sequences in any species and thus enables examination of repeat-prone genomic regions which are thought to be non-informative or ’junk’ sequences. The information non-redundancy of uni-directional RNA sequences compared to information redundancy of bi-directional DNA is demonstrated, a fact that is overlooked by most pattern discovery algorithms. A novel method for computing upstream and downstream miRNA gene boundaries based on mathematical/statistical functions is suggested, as well as cutoffs for annotation of miRNA genes in different miRNA families. Another tool is proposed to allow hypotheses generation and visualization of data matrices, intra- and inter-species chromosomal distribution of miRNA genes or miRNA families. Our results indicate that: miRNA and miRNA

    4. ncRNA-class Web Tool: Non-coding RNA feature extraction and pre-miRNA classification web tool

      KAUST Repository

      Kleftogiannis, Dimitrios A.; Theofilatos, Konstantinos A.; Papadimitriou, Stergios; Tsakalidis, Athanasios K.; Likothanassis, Spiridon D.; Mavroudi, Seferina P.

      2012-01-01

      Until recently, it was commonly accepted that most genetic information is transacted by proteins. Recent evidence suggests that the majority of the genomes of mammals and other complex organisms are in fact transcribed into non-coding RNAs (ncRNAs), many of which are alternatively spliced and/or processed into smaller products. Non coding RNA genes analysis requires the calculation of several sequential, thermodynamical and structural features. Many independent tools have already been developed for the efficient calculation of such features but to the best of our knowledge there does not exist any integrative approach for this task. The most significant amount of existing work is related to the miRNA class of non-coding RNAs. MicroRNAs (miRNAs) are small non-coding RNAs that play a significant role in gene regulation and their prediction is a challenging bioinformatics problem. Non-coding RNA feature extraction and pre-miRNA classification Web Tool (ncRNA-class Web Tool) is a publicly available web tool ( http://150.140.142.24:82/Default.aspx ) which provides a user friendly and efficient environment for the effective calculation of a set of 58 sequential, thermodynamical and structural features of non-coding RNAs, plus a tool for the accurate prediction of miRNAs. © 2012 IFIP International Federation for Information Processing.

    5. Identification of RNA species in the RNA-toxin complex and structure of the complex in Clostridium botulinum type E.

      Science.gov (United States)

      Kitamura, Masaru

      2002-02-15

      Clostridium botulinum type E toxin was isolated in the form of a complex with RNA(s) from bacterial cells. Characterization of the complexed RNA remains to be elucidated. The RNA is identified here as ribosomal RNA (rRNA) having 23S and 16S components. The RNA-toxin complexes were found to be made up of three types with different molecular sizes. The three types of RNA-toxin complex are toxin bound to both the 23S and 16S rRNA, toxin bound to the 16S rRNA and a small amount of 23S rRNA, and toxin bound only to the 16S rRNA. ©2002 Elsevier Science (USA).

    6. Vitamin D and alternative splicing of RNA.

      Science.gov (United States)

      Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

      2015-04-01

      The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

    7. A novel TBP-TAF complex on RNA polymerase II-transcribed snRNA genes.

      Science.gov (United States)

      Zaborowska, Justyna; Taylor, Alice; Roeder, Robert G; Murphy, Shona

      2012-01-01

      Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

    8. RNA precursor pool metabolism and RNA synthesis in X-irradiated Tetrahymena

      International Nuclear Information System (INIS)

      Stephens, R.E.; Paul, I.J.; Zimmerman, A.M.

      1976-01-01

      The incorporation of a radioactive RNA precursor ( 3 H-uridine) has been used in many studies as an index for measuring the synthesis of RNA, yet there is a distinct possibility that the results so obtained were significantly influenced by radiation-induced effects on the metabolism of this precursor into UTP (the primary immediate precursor of RNA) before its incorporation into RNA. A direct examination was therefore undertaken of the effects of X-irradiation on the metabolism of 3 H-uridine and its relationship to RNA synthesis as determined by incorporation. X-irradiation of logarithmically growing Tetrahymena pyriformis caused a dose-dependent depression of total cellular RNA synthesis. Ribosomal RNA (which comprises about 80 per cent of total cellular RNA) synthesis was also depressed by X-irradiation in a dose-dependent manner. Measurements of the levels of radioactivity present in the UTP precursor pool of both irradiated and unirradiated cells were obtained by means of DEAE-cellulose column chromatography of the extracted free nucleotides. Metabolism of 3 H-uridine into UMP, UDP and UTP was depressed by 40%, 26% and 27% respectively, whereas incorporation of 3 H-uridine into RNA was depressed by 77%. The results show that about one-third of the observed (apparent) depression in RNA synthesis was due to radiation-induced effects on the precursor pool, and the remaining two-thirds due to some definite effect of radiation at the transcription level leading to depressed synthesis of RNA. (U.K.)

    9. Extraction of low molecular weight RNA from Citrus trifolita tissues ...

      African Journals Online (AJOL)

      Jane

      2010-12-20

      Dec 20, 2010 ... A critical prerequisite in miRNA studies is acquisition of high quality LMW RNA. LMW RNA is ..... air-dried for a few minutes and then exposed to BIOMAX X-ray film for 48 h using an .... Approaches to microRNA discovery. Nat.

    10. RAIN: RNA-protein Association and Interaction Networks

      DEFF Research Database (Denmark)

      Junge, Alexander; Refsgaard, Jan Christian; Garde, Christian

      2017-01-01

      is challenging due to data heterogeneity. Here, we present a database of ncRNA-RNA and ncRNA-protein interactions and its integration with the STRING database of protein-protein interactions. These ncRNA associations cover four organisms and have been established from curated examples, experimental data...

    11. Interaction of sulforaphane with DNA and RNA.

      Directory of Open Access Journals (Sweden)

      Farzaneh Abassi Joozdani

      Full Text Available Sulforaphane (SFN is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN-DNA and -RNA complexes by Fourier transform infrared (FTIR and UV-Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2, while RNA binding is through G, U, A bases with some degree of SFN-phosphate (PO2 interaction. Overall binding constants were estimated to be K(SFN-DNA=3.01 (± 0.035×10(4 M(-1 and K(SFN-RNA= 6.63 (±0.042×10(3 M(-1. At high SFN concentration (SFN/RNA = 1/1, DNA conformation changed from B to A occurred, while RNA remained in A-family structure.

    12. tmRDB (tmRNA database)

      DEFF Research Database (Denmark)

      Zwieb, Christian; Gorodkin, Jan; Knudsen, Bjarne

      2003-01-01

      Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.html with mirror sites located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Bioinforma......Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.html with mirror sites located at Auburn University, Auburn, Alabama (http......://www.ag.auburn.edu/mirror/tmRDB/) and the Bioinformatics Research Center, Aarhus, Denmark (http://www.bioinf.au.dk/tmRDB/). The tmRDB collects and distributes information relevant to the study of tmRNA. In trans-translation, this molecule combines properties of tRNA and mRNA and binds several proteins to form the tmRNP. Related RNPs are likely...

    13. Accelerated probabilistic inference of RNA structure evolution

      Directory of Open Access Journals (Sweden)

      Holmes Ian

      2005-03-01

      Full Text Available Abstract Background Pairwise stochastic context-free grammars (Pair SCFGs are powerful tools for evolutionary analysis of RNA, including simultaneous RNA sequence alignment and secondary structure prediction, but the associated algorithms are intensive in both CPU and memory usage. The same problem is faced by other RNA alignment-and-folding algorithms based on Sankoff's 1985 algorithm. It is therefore desirable to constrain such algorithms, by pre-processing the sequences and using this first pass to limit the range of structures and/or alignments that can be considered. Results We demonstrate how flexible classes of constraint can be imposed, greatly reducing the computational costs while maintaining a high quality of structural homology prediction. Any score-attributed context-free grammar (e.g. energy-based scoring schemes, or conditionally normalized Pair SCFGs is amenable to this treatment. It is now possible to combine independent structural and alignment constraints of unprecedented general flexibility in Pair SCFG alignment algorithms. We outline several applications to the bioinformatics of RNA sequence and structure, including Waterman-Eggert N-best alignments and progressive multiple alignment. We evaluate the performance of the algorithm on test examples from the RFAM database. Conclusion A program, Stemloc, that implements these algorithms for efficient RNA sequence alignment and structure prediction is available under the GNU General Public License.

    14. A comparison of RNA folding measures

      DEFF Research Database (Denmark)

      Freyhult, E.; Gardner, P. P.; Moulton, V.

      2005-01-01

      the behaviour of these measures over a large range of Rfam ncRNA families. Such measures can be useful in, for example, identifying novel ncRNAs, and indicating the presence of alternate RNA foldings. Results Our analysis shows that ncRNAs, but not mRNAs, in general have lower minimal free energy (MFE) than....... Conclusion Due to the correlations between the different measures we find that it is sufficient to use only two of them in RNA folding studies, one to test if the sequence in question has lower energy than a random sequence with the same dinucleotide frequency (the Z-score) and the other to see......Background In the last few decades there has been a great deal of discussion concerning whether or not noncoding RNA sequences (ncRNAs) fold in a more well-defined manner than random sequences. In this paper, we investigate several existing measures for how well an RNA sequence folds, and compare...

    15. Double Stranded RNA in Human Seminal Plasma

      Directory of Open Access Journals (Sweden)

      Maxim V. Zagoskin

      2017-10-01

      Full Text Available Recently, human semen was shown to contain cell-free nucleic acids, such as DNA, long single stranded RNA, and small RNAs–miRNA and piRNA. The RNAs have been suggested to have potential biological roles as communication molecules between cells and in the temporal and spatial regulation of gene expression in the male reproductive system. Here we demonstrate that human seminal plasma contains a variety of cell-free dsRNAs, describe a robust method to isolate this type of nucleic acid in preparative amounts, and discuss the potential biological roles of these molecules in inheritance. dsRNA plays a role in a variety of biological processes, including gene regulation, is extremely stable and can gain access to cells from the extracellular medium. We suggest that one of the possible functions of dsRNA in human seminal plasma may be to influence human oocytes and therefore, influence the offspring. It also remains possible that these dsRNAs might have potential use as biomarkers for the study of human physiopathological conditions and genetic variation.

    16. A comparison of RNA folding measures

      Directory of Open Access Journals (Sweden)

      Gardner Paul P

      2005-10-01

      Full Text Available Abstract Background In the last few decades there has been a great deal of discussion concerning whether or not noncoding RNA sequences (ncRNAs fold in a more well-defined manner than random sequences. In this paper, we investigate several existing measures for how well an RNA sequence folds, and compare the behaviour of these measures over a large range of Rfam ncRNA families. Such measures can be useful in, for example, identifying novel ncRNAs, and indicating the presence of alternate RNA foldings. Results Our analysis shows that ncRNAs, but not mRNAs, in general have lower minimal free energy (MFE than random sequences with the same dinucleotide frequency. Moreover, even when the MFE is significant, many ncRNAs appear to not have a unique fold, but rather several alternative folds, at least when folded in silico. Furthermore, we find that the six investigated measures are correlated to varying degrees. Conclusion Due to the correlations between the different measures we find that it is sufficient to use only two of them in RNA folding studies, one to test if the sequence in question has lower energy than a random sequence with the same dinucleotide frequency (the Z-score and the other to see if the sequence has a unique fold (the average base-pair distance, D.

    17. Multiple Regression Analysis of mRNA-miRNA Associations in Colorectal Cancer Pathway

      Science.gov (United States)

      Wang, Fengfeng; Wong, S. C. Cesar; Chan, Lawrence W. C.; Cho, William C. S.; Yip, S. P.; Yung, Benjamin Y. M.

      2014-01-01

      Background. MicroRNA (miRNA) is a short and endogenous RNA molecule that regulates posttranscriptional gene expression. It is an important factor for tumorigenesis of colorectal cancer (CRC), and a potential biomarker for diagnosis, prognosis, and therapy of CRC. Our objective is to identify the related miRNAs and their associations with genes frequently involved in CRC microsatellite instability (MSI) and chromosomal instability (CIN) signaling pathways. Results. A regression model was adopted to identify the significantly associated miRNAs targeting a set of candidate genes frequently involved in colorectal cancer MSI and CIN pathways. Multiple linear regression analysis was used to construct the model and find the significant mRNA-miRNA associations. We identified three significantly associated mRNA-miRNA pairs: BCL2 was positively associated with miR-16 and SMAD4 was positively associated with miR-567 in the CRC tissue, while MSH6 was positively associated with miR-142-5p in the normal tissue. As for the whole model, BCL2 and SMAD4 models were not significant, and MSH6 model was significant. The significant associations were different in the normal and the CRC tissues. Conclusion. Our results have laid down a solid foundation in exploration of novel CRC mechanisms, and identification of miRNA roles as oncomirs or tumor suppressor mirs in CRC. PMID:24895601

    18. mRNA/microRNA gene expression profile in microsatellite unstable colorectal cancer

      Directory of Open Access Journals (Sweden)

      Calin George A

      2007-08-01

      Full Text Available Abstract Background Colorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome. Results We investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H for genome-wide expression of microRNA (miRNA and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response. Conclusion This is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

    19. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

      Directory of Open Access Journals (Sweden)

      Kiwamu Hyodo

      2015-05-01

      Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

    20. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

      DEFF Research Database (Denmark)

      Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten

      1994-01-01

      Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows...

    1. A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

      Science.gov (United States)

      Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

      2015-01-29

      Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

    2. RNA damage in biological conflicts and the diversity of responding RNA repair systems

      Science.gov (United States)

      Burroughs, A. Maxwell; Aravind, L.

      2016-01-01

      RNA is targeted in biological conflicts by enzymatic toxins or effectors. A vast diversity of systems which repair or ‘heal’ this damage has only recently become apparent. Here, we summarize the known effectors, their modes of action, and RNA targets before surveying the diverse systems which counter this damage from a comparative genomics viewpoint. RNA-repair systems show a modular organization with extensive shuffling and displacement of the constituent domains; however, a general ‘syntax’ is strongly maintained whereby systems typically contain: a RNA ligase (either ATP-grasp or RtcB superfamilies), nucleotidyltransferases, enzymes modifying RNA-termini for ligation (phosphatases and kinases) or protection (methylases), and scaffold or cofactor proteins. We highlight poorly-understood or previously-uncharacterized repair systems and components, e.g. potential scaffolding cofactors (Rot/TROVE and SPFH/Band-7 modules) with their respective cognate non-coding RNAs (YRNAs and a novel tRNA-like molecule) and a novel nucleotidyltransferase associating with diverse ligases. These systems have been extensively disseminated by lateral transfer between distant prokaryotic and microbial eukaryotic lineages consistent with intense inter-organismal conflict. Components have also often been ‘institutionalized’ for non-conflict roles, e.g. in RNA-splicing and in RNAi systems (e.g. in kinetoplastids) which combine a distinct family of RNA-acting prim-pol domains with DICER-like proteins. PMID:27536007

    3. Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach.

      Science.gov (United States)

      Cambronne, Xiaolu A; Shen, Rongkun; Auer, Paul L; Goodman, Richard H

      2012-12-11

      Identifying targets is critical for understanding the biological effects of microRNA (miRNA) expression. The challenge lies in characterizing the cohort of targets for a specific miRNA, especially when targets are being actively down-regulated in miRNA- RNA-induced silencing complex (RISC)-messengerRNA (mRNA) complexes. We have developed a robust and versatile strategy called RISCtrap to stabilize and purify targets from this transient interaction. Its utility was demonstrated by determining specific high-confidence target datasets for miR-124, miR-132, and miR-181 that contained known and previously unknown transcripts. Two previously unknown miR-132 targets identified with RISCtrap, adaptor protein CT10 regulator of kinase 1 (CRK1) and tight junction-associated protein 1 (TJAP1), were shown to be endogenously regulated by miR-132 in adult mouse forebrain. The datasets, moreover, differed in the number of targets and in the types and frequency of microRNA recognition element (MRE) motifs, thus revealing a previously underappreciated level of specificity in the target sets regulated by individual miRNAs.

    4. Orotidine-Containing RNA: Implications for the Hierarchical Selection (Systems Chemistry Emergence) of RNA.

      Science.gov (United States)

      Kim, Eun-Kyong; Martin, Vincent; Krishnamurthy, Ramanarayanan

      2017-09-12

      The prebiotic synthesis of canonical nucleobases from HCN is a cornerstone for the RNA world hypothesis. However, their role in the primordial pathways to RNA is still debated. The very same process starting from HCN also gives rise to orotic acid, which (via orotidine) plays a crucial role in extant biology in the de novo synthesis of uridine and cytidine, the informational base-pairs in RNA. However, orotidine itself is absent in RNA. Given the prebiotic and biological relevance of orotic acid vis-à-vis uracil, we investigated orotidine-containing RNA oligonucleotides and show that they have severely compromised base-pairing properties. While not unexpected, these results suggest that the emergence of extant RNA cannot just be a consequence of the plausible prebiotic formation of its chemical constituents/building blocks. In combination with other investigations on alternative prebiotic nucleobases, sugars, and linkers, these findings imply that the selection of the components of extant RNA occurred at a higher hierarchical level of an oligomer/polymer based on its functional properties-pointing to a systems chemistry emergence of RNA from a library of precursors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

    5. Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase

      Czech Academy of Sciences Publication Activity Database

      Hashimi, Hassan; Číčová, Zdeňka; Novotná, Lucie; Wen, Y.-Z.; Lukeš, Julius

      2009-01-01

      Roč. 15, č. 4 (2009), s. 588-599 ISSN 1355-8382 R&D Projects: GA ČR GA204/09/1667; GA AV ČR IAA500960705; GA MŠk LC07032; GA MŠk 2B06129; GA ČR GD524/03/H133 Institutional research plan: CEZ:AV0Z60220518 Keywords : RNA editing * guide RNA * mitochondrion * trypanosome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.198, year: 2009

    6. Reproducible pattern of microRNA in normal human skin

      DEFF Research Database (Denmark)

      Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

      2010-01-01

      RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease....

    7. Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

      DEFF Research Database (Denmark)

      Hansen, M.C.; Nielsen, A.K.; Molin, Søren

      2001-01-01

      obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes...

    8. Reproducible pattern of microRNA in normal human skin

      DEFF Research Database (Denmark)

      Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

      2010-01-01

      RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease.......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...

    9. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

      Directory of Open Access Journals (Sweden)

      Kim Chan-Mi

      2007-07-01

      Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

    10. Cancer-Related Triplets of mRNA-lncRNA-miRNA Revealed by Integrative Network in Uterine Corpus Endometrial Carcinoma

      Directory of Open Access Journals (Sweden)

      Chenglin Liu

      2017-01-01

      Full Text Available The regulation of transcriptome expression level is a complex process involving multiple-level interactions among molecules such as protein coding RNA (mRNA, long noncoding RNA (lncRNA, and microRNA (miRNA, which are essential for the transcriptome stability and maintenance and regulation of body homeostasis. The availability of multilevel expression data enables a comprehensive view of the regulatory network. In this study, we analyzed the coding and noncoding gene expression profiles of 301 patients with uterine corpus endometrial carcinoma (UCEC. A new method was proposed to construct a genome-wide integrative network based on variance inflation factor (VIF regression method. The cross-regulation relations of mRNA, lncRNA, and miRNA were then selected based on clique-searching algorithm from the network, when any two molecules of the three were shown as interacting according to the integrative network. Such relation, which we call the mRNA-lncRNA-miRNA triplet, demonstrated the complexity in transcriptome regulation process. Finally, six UCEC-related triplets were selected in which the mRNA participates in endometrial carcinoma pathway, such as CDH1 and TP53. The multi-type RNAs are proved to be cross-regulated as to each of the six triplets according to literature. All the triplets demonstrated the association with the initiation and progression of UCEC. Our method provides a comprehensive strategy for the investigation of transcriptome regulation mechanism.

    11. Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells.

      Science.gov (United States)

      Alshehri, Abdullah; Grabowska, Anna; Stolnik, Snow

      2018-02-28

      Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the l