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Sample records for largest ribonucleoprotein particle

  1. X-ray Crystal Structure of the Vault, Largest Ribonucleoprotein Particle, with a Molecular Weight of 10 MDa

    NASA Astrophysics Data System (ADS)

    Tanaka, Hideaki; Kato, Koji; Yamashita, Eiki

    Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Although roles in multidrug resistance and innate immunity have been suggested, the cellular function remains unclear. We have determined the X-ray structure of rat liver vault at 3.5 Å resolution. A vault particle shell was composed of 78 MVP (Major vault protein) chains with 39-fold dihedral symmetry. The shoulder domain of MVP is structurally similar to SPFH (stomatin/prohibitin/flotillin/HflK/C) domain involved in lipid raft association.

  2. Axonal transport of ribonucleoprotein particles (vaults).

    PubMed

    Li, J Y; Volknandt, W; Dahlstrom, A; Herrmann, C; Blasi, J; Das, B; Zimmermann, H

    1999-01-01

    RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon

  3. Structural Dynamics of the Vault Ribonucleoprotein Particle

    NASA Astrophysics Data System (ADS)

    Casañas, Arnau; Querol, Jordi; Fita, Ignasi; Verdaguer, Núria

    Vaults are ubiquitous, highly conserved, 13 MDa ribonucleoprotein particles, involved in a diversity of cellular processes, including multidrug resistance, transport mechanisms and signal transmission. There are between 104 and 106 vault particles per mammalian cell and they do not trigger autoimmunity. The vault particle shows a hollow barrel-shaped structure organized in two identical moieties, each consisting of 39 copies of the major vault protein (MVP). Other data indicated that vault halves can dissociate at acidic pH. The high resolution, crystal structure of the of the seven N-terminal domains (R1-R7) of MVP, forming the central vault barrel, together with that of the native vault particle (solved at 8 Å resolution), revealed the interactions governing vault association and suggested a pH-dependent mechanism for a reversible dissociation induced by low pH. Vault particles posses many features making them very promising vehicles for the delivery of therapeutic agents including self-assembly, 100 nm size range, emerging atomic-level structural information, natural presence in humans ensuring biocompability, recombinant production system, existing features for targeting species to the large lumen and a dynamic structure that may be controlled for manipulation of drug release kinetics. All these attributes provide vaults with enormous potential as a drug/gene delivery platform.

  4. A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry

    SciTech Connect

    Kato, Koji; Tanaka, Hideaki; Sumizawa, Tomoyuki; Yoshimura, Masato; Yamashita, Eiki; Iwasaki, Kenji; Tsukihara, Tomitake

    2008-05-01

    A vault from rat liver was crystallized in space group C2. Rotational symmetry searches indicated that the particle has 39-fold dihedral symmetry. Vault is a 12.9 MDa ribonucleoprotein particle with a barrel-like shape, two protruding caps and an invaginated waist structure that is highly conserved in a wide variety of eukaryotes. Multimerization of the major vault protein (MVP) is sufficient to assemble the entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1), as well as a small vault RNA (vRNA), are also associated with vault. Here, the crystallization of vault particles is reported. The crystals belong to space group C2, with unit-cell parameters a = 708.0, b = 385.0, c = 602.9 Å, β = 124.8°. Rotational symmetry searches based on the R factor and correlation coefficient from noncrystallographic symmetry (NCS) averaging indicated that the particle has 39-fold dihedral symmetry.

  5. Vaults: a ribonucleoprotein particle involved in drug resistance?

    PubMed

    Mossink, Marieke H; van Zon, Arend; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2003-10-20

    Vaults are ribonucleoprotein particles found in the cytoplasm of eucaryotic cells. The 13 MDa particles are composed of multiple copies of three proteins: an M(r) 100 000 major vault protein (MVP) and two minor vault proteins of M(r) 193 000 (vault poly-(ADP-ribose) polymerase) and M(r) 240 000 (telomerase-associated protein 1), as well as small untranslated RNA molecules of approximately 100 bases. Although the existence of vaults was first reported in the mid-1980s no function has yet been attributed to this organelle. The notion that vaults might play a role in drug resistance was suggested by the molecular identification of the lung resistance-related (LRP) protein as the human MVP. MVP/LRP was found to be overexpressed in many chemoresistant cancer cell lines and primary tumor samples of different histogenetic origin. Several, but not all, clinico-pathological studies showed that MVP expression at diagnosis was an independent adverse prognostic factor for response to chemotherapy. The hollow barrel-shaped structure of the vault complex and its subcellular localization indicate a function in intracellular transport. It was therefore postulated that vaults contributed to drug resistance by transporting drugs away from their intracellular targets and/or the sequestration of drugs. Here, we review the current knowledge on the vault complex and critically discuss the evidence that links vaults to drug resistance. PMID:14576851

  6. Studies on dissociation and reconstitution of nuclear 30-S ribonucleoprotein particles containing pre-mRNA.

    PubMed

    Kulguskin, V V; Krichevskaya, A A; Lukanidin, E M; Georgiev, G P

    1980-10-17

    Treatment of nuclear 30-S ribonucleoprotein (RNP) particles containing pre-mRNA (precursor of mRNA) with 2 M NaCl leads to dissociation of RNA and protein. The protein component is present either as an aggregate with a sedimentation coefficient close to 30 S (a free informofer) or as a slowly sedimenting material (monomers or oligomers of informatin). Most of the informofers and slowly sedimenting material are in the equilibrium state. Iodination or aging of the 30-S particles stabilizes informofers. Lowering of NaCl concentration in the mixture of RNA with informofers or informatin subunits leads to reconstitution of RNP particles. In both cases, the particles formed have a sedimentation coefficient of about 30 S and a buoyant density equal to 1.4-1.41 g/cm3 but their response to pancreatic RNAase (EC 3.1.27.5) and high salt treatment is very different. Both the particles reconstituted from RNA and informofers and the original particles are very sensitive to pancreatic RNAase and after high salt treatment free informofers are formed. In contrast, the RNA of the particles reconstituted from slowly sedimenting material is much more protected against pancreatic RNAase action. These particles are also rather stable to high salt treatment. Thus, only if a protein in the form of an informofer aggregate is used, faithful reconstitution takes place. The data obtained are discussed in terms of the structure of the nuclear ribonucleoprotein particles containing precursor of messenger RNA. PMID:7437433

  7. The ribonucleoprotein nature of large particles in the meiosporangia of Allomyces.

    PubMed

    ROREM, E S; MACHLIS, L

    1957-11-25

    Particles averaging 3 to 4 micro in diameter, which are called chromospheres and fill the immature meiosporangia of the watermold Allomyces, were isolated and analyzed. The preparations were obtained by repeated centrifugations or by passage of the homogenate into a column of sand saturated with oleic acid, followed by selective elution of the chromospheres with alternate layers of oleic acid and an aqueous solution. The chromospheres contain approximately 12 per cent RNA, no DNA, and 60 per cent protein. It was concluded that they are pure or nearly pure ribonucleoprotein. Analysis of meiosporangia with chromospheres and after the chromospheres have disappeared showed no significant change in RNA or free amino acids. It was concluded that chromosphere disappearance is a fragmentation into small granules. The relation of chromospheres to postmeiotic chromospheres and nuclear caps is discussed. Speculation as to the function of these bodies is presented. PMID:13481022

  8. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities

    SciTech Connect

    Swanson, M.S.; Dreyfuss, G.

    1988-05-01

    Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. The authors show that the hnRNP proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.

  9. Characterization of messenger ribonucleoprotein particles in dormant sporangiospores of the fungus Mucor racemosus

    SciTech Connect

    Chapman, C.P.

    1986-01-01

    Extracts of sporangiospores of Mucor racemosus contained RNA that readily hybridized with (/sup 3/H)polyuridylic acid. Prior to germination, this RNA was in a form sedimenting at <80S. Within 10 minutes after initiating germination, most of this RNA sedimented with polyribosomes and 80S monoribosomes. Particulate material from spore extracts bound to oligo(dT)-cellulose at high ionic strength and was assumed to contain messenger ribonucleoprotein particles (mRNP's). A portion of the mRNP's was released from the column by lowering the ionic strength. Other portions were eluted stepwise in buffer containing 50% and 90% formamide and in 0.1-N NaOH. Identical elution patterns were observed whether monitoring incorporated /sup 31/P-orthophosphate or L-(/sup 32/S)methionine, absorbance at 280 nm, or hybridization of (/sup 3/H)polyuridylic acid. mRNP's from the first two fractions were analyzed. A bimodal population of particles was detected in sedimentation velocity and sedimentation equilibrium centrifugation. Particles eluted at low ionic strength demonstrated a sedimentation coefficient distribution of 20S-to-80S. Particles eluted in formamide demonstrated a sedimentation coefficient distribution of 20S-to-60S. Particles eluted at low ionic strength displayed two peaks in CsCl centrifugation, with buoyant densities of 1.37 gm/cc and 1.59 gm/cc. Particles eluted in formamide displayed a single peak with a buoyant density of 1.61 gm/cc. Particles eluted at low ionic strength and centrifuged in metrizamide solution formed two bands having buoyant densities of 1.15 gm/cc and 1.30 gm/cc; formamide-eluted particles banded only at the higher density. Mucor 40S ribosomal subunits banded at 1.56 gm/cc and 1.28 gm/cc in CsCl and metrizamide solution respectively.

  10. Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry.

    PubMed

    Kedersha, N L; Heuser, J E; Chugani, D C; Rome, L H

    1991-01-01

    The structure of rat liver vault ribonucleoprotein particles was examined using several different staining techniques in conjunction with EM and digestion with hydrolytic enzymes. Quantitative scanning transmission EM demonstrates that each vault particle has a total mass of 12.9 +/- 1 MD and contains two centers of mass, suggesting that each vault particle is a dimer. Freeze-etch reveals that each vault opens into delicate flower-like structures, in which eight rectangular petals are joined to a central ring, each by a thin hook. Vaults examined by negative stain and conventional transmission EM (CTEM) also reveal the flower-like structure. Trypsin treatment of vaults resulted exclusively in cleavage of the major vault protein (p104) and concurrently alters their structure as revealed by negative stain/CTEM, consistent with a localization of p104 to the flower petals. We propose a structural model that predicts the stoichiometry of vault proteins and RNA, defines vault dimer-monomer interactions, and describes two possible modes for unfolding of vaults into flowers. These highly dynamic structural variations are likely to play a role in vault function. PMID:1988458

  11. Functional and structural consequences of uridine substitutions on H/ACA ribonucleoprotein particle pseudouridine synthase

    PubMed Central

    Zhou, Jing; Liang, Bo; Li, Hong

    2010-01-01

    Box H/ACA ribonucleoprotein protein particles catalyze the majority of pseudouridylation in functional RNA. Different from stand alone pseudouridine synthases, the RNP pseoduridine synthase comprises multiple protein and an RNA subunits. Previous studies showed that each subunit, regardless its location, is sensitive to the step of subunit placement at the catalytic center and potentially to the reaction status of the substrate. Here we describe the impact of chemical substitutions of target uridine on enzyme activity and structure. We found that 3-methyluridine in place of uridine inhibited its isomerization while 2’-deoxyuridine or 4-thiouridine did not. Significantly, crystal structures of an archaeal box H/ACA RNP bound with the nonreactive and the two post-reactive substrate analogs showed only subtle structural changes throughout the assembly except for a conserved tyrosine and a substrate anchoring loop of Cbf5. Our results suggest a potential role of these elements and the subunit that contacts them in substrate binding and product release. PMID:20575532

  12. Structural Basis for Substrate Placement by an Archaeal Box C/D Ribonucleoprotein Particle

    SciTech Connect

    Xue, Song; Wang, Ruiying; Yang, Fangping; Terns, Rebecca M.; Terns, Michael P.; Zhang, Xinxin; Maxwell, E.Stuart; Li, Hong

    2012-05-09

    Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2'-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs.

  13. Structural basis for substrate placement by an archaeal box C/D ribonucleoprotein particle

    PubMed Central

    Xue, Song; Wang, Ruiying; Yang, Fangping; Terns, Rebecca M.; Terns, Michael P.; Zhang, Xinxin; Maxwell, E. Stuart; Li, Hong

    2012-01-01

    Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2’-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a halfmer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modelling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and that methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs. PMID:20864039

  14. Stage-specific synthesis of proteins complexed to ribonucleoprotein particles and ribosomes in zoospores of Blastocladiella emersonii.

    PubMed

    Jaworski, A J; Stumhofer, P

    1981-04-01

    In Blastocladiella emersonii zoospores, a set of proteins was found associated with the ribosomes and free ribonucleoprotein particles distinct from the ribosomes and polyribosomes. These proteins were designated P120, P105, P64, P56, and P42 based on their molecular weights determined by gel electrophoresis. Synthesis of these proteins was detected only during late sporulation just before the time polyadenylated ribonucleic acid accumulates in the sporangia. These proteins banded in isopycnic metrizamide gradients at densities of 1.31 and 1.27 g/cm3, which corresponded to the densities of the ribosomes and free ribonucleoprotein particles, respectively. Comparison of the distribution of the proteins in sucrose versus metrizamide gradients suggested that P105 was removed from the free ribonucleoprotein particles before complexing with the ribosomes. During germination, these proteins disappeared from the ribosomal fractions, with kinetics corresponding to the resumption of protein synthesis. Another protein (P178) was observed to bind to the ribosomes before the onset of protein synthesis during germination. Cycloheximide did not block the addition of this protein to the monoribosomes. PMID:6086010

  15. Preliminary analysis of two and three dimensional crystals of vault ribonucleoprotein particles.

    PubMed

    Querol-Audí, Jordi; Perez-Luque, Rosa; Fita, Ignacio; Lopéz-Iglesias, Carmen; Castón, José R; Carrascosa, José L; Verdaguer, Nuria

    2005-07-01

    Vaults are large ribonucleoprotein particles found in a wide variety of eukaryotes. When imaged by electron-microscopy vaults present a strikingly conserved barrel-shaped structure with an invaginated waist and two protruding caps. In this work, we present two dimensional (2D) and three dimensional (3D) crystals of naturally produced vaults in murine and monkey cells, respectively. The 2D-crystals presented a hexagonal packing with the lattice parameter defined by the diameter of the vault barrel. Fourier transforms from images of the negatively stained 2D-crystals showed spots till about 45 A resolution. The 3D-crystals reached about 0.15 x 0.15 x 0.02 mm3 in size and presented a flat triangular morphology with well-developed faces. The preliminary characterization of these 3D-crystals, which diffract very weakly to approximately 10 A resolution, suggests a trigonal packing with the R32 space group symmetry. The 3D-crystals appear to be formed by adding layers of vaults, which retain the hexagonal organization seen in the 2D-crystals, with relative shifts that maximize the interdigitation of particles in adjacent layers. Accurate crystal symmetry in the 2D- and 3D-crystals requires neighbor particles interacting according to a 6-fold and a 3-fold dihedral symmetry, respectively. Compatibility with the reported 8-fold symmetry would imply multiples of 24-fold rotational symmetry, in agreement with the recently proposed 48-fold dihedral symmetry for reconstituted recombinant vaults. PMID:15964767

  16. Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles.

    PubMed Central

    Wurtz-T; Kiseleva, E; Nacheva, G; Alzhanova-Ericcson, A; Rosén, A; Daneholt, B

    1996-01-01

    Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the particles. Thus, the 36- and 45-KDa proteins are likely to be abundant, RNA-binding proteins in the BR particles. To elucidate to what extent the two proteins are also present in other hnRNPs, we studied the binding of the antibodies to chromosomal puffs in general. It was observed that many puffs in addition to the BRs harbor the two proteins, but there are also puffs containing only one of the components, either the 36- or the 45-kDa protein. We conclude that the two proteins are not randomly bound to all hnRNPs but that each of them seems to be linked to a specific subset of the particles. PMID:8657116

  17. The 3.2 Angstrom Resolution Structure of the Polymorphic Cowpea Chlorotic Mottle Virus Ribonucleoprotein Particle

    NASA Astrophysics Data System (ADS)

    Speir, Jeffrey Alan

    Structural studies of the polymorphic cowpea chlorotic mottle virus have resulted in high resolution structures for two distinct icosahedral ribonucleoprotein particle conformations dependent upon whether acidic or basic pH conditions prevail. CCMV is stable below pH 6.5, however metal-free particles maintain a 10% increase in hydrodynamic volume at pH >=q 7.5. Identification of this swollen' form of CCMV, which can easily be disrupted with 1M NaCl, led to the first reassembly of an icosahedral virus in vitro from purified viral protein and RNA to form infectious particles, and its assembly has been the subject of biochemical and biophysical investigations for over twenty-five years. Under well defined conditions of pH, ionic strength and divalent metal ion concentration, CCMV capsid protein or capsid protein and RNA will reassemble to form icosahedral particles of various sizes, sheets, tubes, rosettes, and a variety of laminar structures which resemble virion structures from non-related virus families. Analysis of native particles at 3.2A resolution and swollen particles at 28A resolution has suggested that the chemical basis for the formation of polymorphic icosahedral and anisometric structures is: (i) hexamers formed of beta-barrel subunits stabilized by an unusual hexameric parallel beta structure made up of their N-termini, (ii) the location of protein-RNA interactions, (iii) divalent metal cation binding sites that regulate quasi-symmetrical subunit associations, (iv) charge repulsion across the same interfaces when lacking divalent metal ions at basic pH, which induces the formation of sixty 20A diameter portals for RNA release, and (v) a novel, C-terminal-based, subunit dimer assembly unit. The use of C- and N-terminal arms in CCMV has not been observed in other icosahedral RNA virus structures determined at near atomic resolution, however, their detailed interactions and roles in stabilizing the quaternary organization of CCMV are related to that found

  18. U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

    PubMed

    Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

    2014-04-25

    The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. PMID:24583175

  19. Translational Control of the Oogenic Program by Components of OMA Ribonucleoprotein Particles in Caenorhabditis elegans

    PubMed Central

    Spike, Caroline A.; Coetzee, Donna; Nishi, Yuichi; Guven-Ozkan, Tugba; Oldenbroek, Marieke; Yamamoto, Ikuko; Lin, Rueyling; Greenstein, David

    2014-01-01

    The oocytes of most sexually reproducing animals arrest in meiotic prophase I. Oocyte growth, which occurs during this period of arrest, enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. In the nematode Caenorhabditis elegans, the major sperm protein hormone promotes meiotic resumption (also called meiotic maturation) and the cytoplasmic flows that drive oocyte growth. Prior work established that two related TIS11 zinc-finger RNA-binding proteins, OMA-1 and OMA-2, are redundantly required for normal oocyte growth and meiotic maturation. We affinity purified OMA-1 and identified associated mRNAs and proteins using genome-wide expression data and mass spectrometry, respectively. As a class, mRNAs enriched in OMA-1 ribonucleoprotein particles (OMA RNPs) have reproductive functions. Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3′-untranslated regions (3′-UTRs). Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2. We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs. In the accompanying article in this issue, we show that LIN-41 prevents meiotic maturation and promotes oocyte growth in opposition to OMA-1/2. Taken together, these data support a model in which the conserved regulators of mRNA translation LIN-41 and OMA-1/2 coordinately control oocyte growth and the proper spatial and temporal execution of the meiotic maturation decision. PMID:25261697

  20. Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

    PubMed

    Sato, T; Ishikawa, K; Ogata, K

    1977-02-16

    The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system. PMID:319832

  1. Fluorescence studies on the role of tryptophan in heterogeneous nuclear ribonucleoprotein particles of HeLa cells.

    PubMed Central

    Schenkel, J; Appel, I; Schwarzwald, R; Bautz, E k; Wolfrum, J; Greulich, K O

    1989-01-01

    The 40 S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from HeLa cells reveal tryptophan fluorescence with a bi-exponential decay, indicating that only a few of the 'core' proteins contain tryptophan residues. The presence of tryptophan residues distinguishes hnRNP particles from nucleosomes, with which they otherwise share a number of properties. This difference, however, is not essential for protein-RNA binding, as the fluorescence decay remains unchanged when hnRNP particles are dissociated into protein and RNA. However, the Stern-Volmer quenching constant is doubled upon salt dissociation, i.e. tryptophan residues become more accessible to solvent. Thus tryptophan quenching is a useful parameter for monitoring protein-protein interactions in hnRNP particles. PMID:2604698

  2. 3'-end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei.

    PubMed Central

    Terns, M P; Lund, E; Dahlberg, J E

    1992-01-01

    We have identified and characterized a U6 small nuclear (sn) ribonucleoprotein particle (RNP) present in the nuclei of Xenopus laevis oocytes. The structure of this U6 snRNP was investigated by native gel shift analysis and a combination of RNA-protein UV cross-linking, RNase T1 fingerprinting, and immunoprecipitation assays. These analyses demonstrate that certain forms of U6 snRNA associate with the 50-kDa nuclear antigen La both in vivo and in vitro. The La protein binds the stretch of uridylates at the 3' hydroxyl end of newly synthesized U6 snRNA. La does not bind to mature U6 snRNAs that have 2',3'-cyclic phosphate (greater than p) groups at their 3' ends (E. Lund and J. E. Dahlberg, Science 255:327-330, 1992) or to U6 snRNAs in anti-Sm-precipitable U4/U6 snRNPs. We propose that 3'-end modification, including posttranscriptional UMP addition, modulates the binding of La protein to U6 snRNA which, in turn, may affect the function of this RNA. Images PMID:1535684

  3. Functional and structural impact of target uridine substitutions on the H/ACA ribonucleoprotein particle pseudouridine synthase.

    PubMed

    Zhou, Jing; Liang, Bo; Li, Hong

    2010-07-27

    Box H/ACA ribonucleoprotein protein particles catalyze the majority of pseudouridylation in functional RNA. Different from stand alone pseudouridine synthases, the RNP pseudouridine synthase comprises multiple protein subunits and an RNA subunit. Previous studies showed that each subunit, regardless its location, is sensitive to the step of subunit placement at the catalytic center and potentially to the reaction status of the substrate. Here we describe the impact of chemical substitutions of target uridine on enzyme activity and structure. We found that 3-methyluridine in place of uridine inhibited its isomerization while 2'-deoxyuridine or 4-thiouridine did not. Significantly, crystal structures of an archaeal box H/ACA RNP bound with the nonreactive and the two postreactive substrate analogues showed only subtle structural changes throughout the assembly except for a conserved tyrosine and a substrate anchoring loop of Cbf5. Our results suggest a potential role of these elements and the subunit that contacts them in substrate binding and product release. PMID:20575532

  4. Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA.

    PubMed

    Kedersha, N L; Rome, L H

    1986-09-01

    Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 X 65 nm and are therefore considerably larger than ribosomes. When subjected to SDS PAGE, these structures, which we refer to as vaults, appear to contain several minor and five major species: Mr 210,000, 192,000, 104,000, 54,000, and 37,000. One of these (Mr 104,000) greatly predominates, accounting for greater than 70% of the total Coomassie Brilliant Blue-staining protein. Another major species of Mr 37,000 has been identified as a species of small RNA of unusual base composition (adenosine 12.0%, guanosine 29.7%, uridine 30.9%, and 27.4% cytidine), which migrates as a single species in urea PAGE between the 5S and 5.8S ribosomal standards, containing approximately 140 bases. Although the RNA constitutes only 4.6% of the entire structure, the large size of the particle requires that each one contains approximately 9 molecules of this RNA. Antibodies prepared against the entire particle are largely specific for the major (Mr 104,000) polypeptide species. Although they do not directly react with the RNA constituent on Western blots, these antibodies immunoprecipitate a 32P-labeled RNA of identical size from metabolically-labeled rat hepatoma cells. Vaults are observed in partially purified fractions from human fibroblasts, murine 3T3 cells, glial cells, and rabbit alveolar macrophages. It therefore appears that these novel ribonucleoprotein structures are broadly distributed among different cell types. The function of vaults is at present unknown. PMID:2943744

  5. Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast

    PubMed Central

    Niedner, Annika; Müller, Marisa; Moorthy, Balaji T.; Jansen, Ralf-Peter; Niessing, Dierk

    2013-01-01

    Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export. The nucleolar localization of ASH1 mRNA protein 1 (Loc1p) is required for efficient ASH1 mRNA localization. Immunoprecipitation experiments have revealed that Loc1p forms cocomplexes with other components of the ASH1 transport complex. However, it remains unclear how Loc1p is recruited into this mRNP and why Loc1p is important for ASH1 mRNA localization. Here we demonstrate that Loc1p undergoes a direct and specific interaction with the ASH1 mRNA-binding Swi5p-dependent HO expression protein 2 (She2p). This cocomplex shows higher affinity and specificity for RNA bearing localization elements than the individual proteins. It also stabilizes the otherwise transient binding of She2p to ASH1 mRNA, suggesting that cooperative mRNA binding of Loc1p with She2p is the required nuclear function of Loc1p for ASH1 mRNA localization. After nuclear export, myosin-bound She3p joins the ASH1 mRNP to form a highly specific cocomplex with She2p and ASH1 mRNA. Because Loc1p is found only in the nucleus, it must be removed from the complex directly before or after export. In vitro and in vivo experiments indicate that the synergistic interaction of She2p and She3p displaces Loc1p from the ASH1 complex, allowing free Loc1p to rapidly reenter the nucle(ol)us. Together these findings suggest an ordered process of nuclear assembly and reorganization for the maturation of localizing ASH1 mRNPs. PMID:24324176

  6. RNA-RNA interaction is required for the formation of specific bicoid mRNA 3' UTR-STAUFEN ribonucleoprotein particles.

    PubMed Central

    Ferrandon, D; Koch, I; Westhof, E; Nüsslein-Volhard, C

    1997-01-01

    The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid (bcd) to the anterior pole of the egg cell. Staufen protein (STAU) is required in a late step of the localization to anchor the bcd mRNA in the anterior cytoplasm. We have shown previously that endogenous STAU associates specifically with injected bcd mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by STAU in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. Here, we show that the STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. In addition, base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intramolecular; thus dimers or multimers of the RNA localization signal must be associated with STAU in these particles. The bcd mRNA 3' UTR can also dimerize in vitro in the absence of STAU. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization. PMID:9130719

  7. Discovery of Nuclear DNA-like RNA (dRNA, hnRNA) and Ribonucleoproteins Particles Containing hnRNA.

    PubMed

    Georgiev, G P

    2016-01-01

    On August 9-11, 2014, Cold Spring Harbor (USA) hosted a special symposium dedicated to the discovery of messenger or informational RNA and the main events in the subsequent studies of its synthesis, regulation of synthesis, maturation, and transport. The existence of mRNA in bacteria was first suggested in 1961 by Jacob and Monod, based on genetic studies [1]. The same year, Brenner et al. confirmed the hypothesis [2]. Our laboratory played a key role in the discovery of messenger RNA in eukaryotes, as well as in the discovery of the nuclear ribonucleoproteins that contain it and in the elucidation of their structural organization. Therefore, I was invited to represent Russia at the Symposium and deliver a speech on these topics. However, my visa had only been issued after the end of the Symposium, and, therefore, the presentation was delivered by my former colleague G.N. Yenikolopov, who works at Cold Spring Harbor Laboratory. The transcript of the lecture is presented below. PMID:27099780

  8. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  9. The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles.

    PubMed Central

    Huang, M; Rech, J E; Northington, S J; Flicker, P F; Mayeda, A; Krainer, A R; LeStourgeon, W M

    1994-01-01

    A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts. Images PMID:8264621

  10. Particle Physics in the Sky and Astrophysics Underground: Connecting the Universe's Largest and Smallest Scales

    NASA Astrophysics Data System (ADS)

    Swanson, Molly E. C.

    2008-08-01

    Particles have tremendous potential as astronomical messengers, and conversely, studying the universe as a whole also teaches us about particle physics. This thesis encompasses both of these research directions. Many models predict a diffuse flux of high energy neutrinos from active galactic nuclei and other astrophysical sources. The "Astrophysics Underground" portion of this thesis describes a search for this neutrino flux performed by looking for very high energy upward-going muons using the Super-K detector. In addition to using particles to do astronomy, we can also use the universe itself as a particle physics lab. The "Particle Physics in the Sky" portion of this thesis focuses on extracting cosmological information from galaxy surveys. To overcome technical challenges faced by the latest galaxy surveys, we produced a comprehensive upgrade to mangle, a software package that processes the angular masks defining the survey area on the sky. We added dramatically faster algorithms and new useful features that are necessary for managing complex masks of current and next-generation galaxy surveys. With this software in hand, we utilized SDSS data to investigate the relation between galaxies and dark matter by studying relative bias, i.e., the relation between different types of galaxies. Separating galaxies by their luminosities and colors reveals a complicated picture: red galaxies are clustered more strongly than blue galaxies, with both the brightest and the faintest red galaxies showing the strongest clustering. Furthermore, red and blue galaxies tend to occupy different regions of space. In order to make precise measurements from the next generation of galaxy surveys, it will be essential to account for this complexity.

  11. Nuclear Ribonucleoprotein Complexes Containing Polyadenylate from Mouse Ascites Cells

    PubMed Central

    Quinlan, Thomas J.; Billings, Peter B.; Martin, Terence E.

    1974-01-01

    Nuclear poly(A)-containing RNA of mouse ascites cells can be extracted in the form of 15-17S ribonucleoprotein complexes under conditions in which the bulk of the heterogeneous nuclear RNA is released as 30S complexes. The poly(A)-containing fraction of nuclear extracts has been resolved into two distinct components, 15 and 17 S; neither contains the two polypeptides of 30S ribonucleoprotein. The 17S particle contains approximately six polypeptide species of molecular masses 17,000-30,000 daltons. The 15S complex has four distinct polypeptides of higher molecular mass, including a prominent 80,000-dalton species. PMID:4368966

  12. Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate.

    PubMed

    van Nues, Rob W; Castro-Roa, Daniel; Yuzenkova, Yulia; Zenkin, Nikolay

    2016-04-01

    Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAPin vivoandin vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins. PMID:26609136

  13. Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate

    PubMed Central

    van Nues, Rob W.; Castro-Roa, Daniel; Yuzenkova, Yulia; Zenkin, Nikolay

    2016-01-01

    Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins. PMID:26609136

  14. Ribonucleoprotein multimers and their functions

    PubMed Central

    Bleichert, Franziska; Baserga, Susan J.

    2010-01-01

    Ribonucleoproteins (RNPs) play key roles in many cellular processes and often function as RNP enzymes. Similar to proteins, some of these RNPs exist and function as multimers, either momomeric or heteromeric. While in some cases the mechanistic function of multimerization is well understood, the functional consequences of multimerization of other RNPs remain enigmatic. In this review we will discuss the function and organization of small RNPs that exist as stable multimers, including RNPs catalyzing RNA chemical modifications, telomerase RNP, and RNPs involved in pre-mRNA splicing. PMID:20572804

  15. Ribonucleoprotein complexes of hepatitis delta virus.

    PubMed Central

    Ryu, W S; Netter, H J; Bayer, M; Taylor, J

    1993-01-01

    Human hepatitis delta virus (HDV) is a subviral satellite agent of hepatitis B virus (HBV). The envelope proteins of HDV are provided by the helper virus, HBV, but very little is known about the internal structure of HDV. The particles contain multiple copies of the delta antigen and an unusual RNA genome that is small, about 1,700 nucleotides in length, single stranded, and circular. By using UV cross-linking, equilibrium density centrifugation, and immunoprecipitation, we obtained evidence consistent with the interpretation that delta antigen and genomic RNA form a stable ribonucleoprotein (RNP) complex within the virion. Furthermore, electron-microscopic examination of the purified viral RNP revealed a roughly spherical core-like structure with a diameter of 18.7 +/- 2.5 nm. We also isolated HDV-specific RNP structures from the nuclei of cells undergoing HDV genome replication; both the genome and antigenome (a complement of the genome) of HDV were found to be in such complexes. From the equilibrium density analyses of the viral and nuclear RNPs, we were able to deduce the number of molecules of delta antigen per molecule of HDV RNA. For virions, this number was predominantly ca. 70, which was larger than for the nuclear RNPs, which were more heterogeneous, with an average value of ca. 30. Images PMID:8497052

  16. Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.

    PubMed Central

    Swanson, M S; Nakagawa, T Y; LeVan, K; Dreyfuss, G

    1987-01-01

    In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative

  17. Phosphorylation of the C proteins in heterogeneous ribonucleoprotein (hnRNP) particles in HeLa cells: Characterization of in vivo phosphorylation, comparison with in vitro phosphorylation using casein kinase II, and preliminary studies on the effects of phosphorylation on particle structure

    SciTech Connect

    Kleiman, N.J.

    1989-01-01

    Newly formed pre-messenger RNA associates with protein to form heterogeneous ribonucleoprotein (hnRNP) particles. In HeLa cells, hnRNP particles contain six core proteins. Two proteins, termed C{sub 1} and C{sub 2}, are phosphorylated in vitro by casein kinase 11 (CKII). C{sub 1} protein became {sup 32}P-labeled after HeLa cells were incubated with ({sup 32}P)-orthophosphate in vivo (ibid). Because phosphorylation is a ubiquitous regulatory mechanism, C protein phosphorylation was studied in greater detail. C protein phosphorylation in hnRNP particles was investigated in HeLa cells incubated with ({sup 32}P)-orthophosphate in vivo. Immunoblotting in pH 3.5-10 isoelectric focusing (IEF) gels indicated that C proteins focus only at pH 5.0. In pH 4.5-5.5 IEF gels, individually purified C, and 2 proteins resolve into the same four closely spaced, {sup 32}P-labeled bands. A fifth, unlabeled, more basic species was detached when hnRNP particles were purified without NaF. All {sup 32}P-labeled species contained identical amounts of {sup 32}P per unit protein suggesting that charge heterogeneity is not due to differential phosphorylation. Attempts to detect bound carbohydrate were unsuccessful. {sup 32}P-labeled phosphate was readily removed by potato acid phosphatase. E. coli alkaline phosphatase and snake venom phosphodiesterase were ineffective. {sup 32}P-label was found exclusively in phosphoserine. One-dimensional peptide mapping with chymotrypsin and S. aureus protease detected two phosphorylated peptides. C protein phosphorylation was also investigated in vitro. Incubation of hnRNP particles with rabbit liver CKII and {sup 32}P-ATP followed by IEF in pH 4.5-5.5 gels indicated that all four C protein species were {sup 32}P-labeled. {sup 32}P-label was found exclusively in phosphoserine.

  18. Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.

    PubMed

    Zheng, Chun-Lei; Sumizawa, Tomoyuki; Che, Xiao-Fang; Tsuyama, Shinichiro; Furukawa, Tatsuhiko; Haraguchi, Misako; Gao, Hui; Gotanda, Takenari; Jueng, Hei-Cheul; Murata, Fusayoshi; Akiyama, Shin-Ichi

    2005-01-01

    Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly. PMID:15567158

  19. Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: Further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells

    SciTech Connect

    Hendrick, J.P.; Wolin, S.L.; Rinke, J.; Lerner, M.R.; Steitz, J.A.

    1981-12-01

    Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5'-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The RoRNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed.

  20. Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection

    PubMed Central

    Jagdeo, Julienne M.; Dufour, Antoine; Fung, Gabriel; Luo, Honglin; Kleifeld, Oded; Overall, Christopher M.

    2015-01-01

    ABSTRACT Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M (hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNP M is cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNP M relocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNP M using small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNP M is a general strategy utilized by picornaviruses to facilitate viral infection. IMPORTANCE Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNP M as a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNP M and redirects it to a step in the viral life cycle. We propose that cleavage of hnRNP M is a general strategy that picornaviruses use to facilitate infection. PMID:25926642

  1. Largest College Endowments, 2011

    ERIC Educational Resources Information Center

    Chronicle of Higher Education, 2012

    2012-01-01

    Of all endowments valued at more than $250-million, the UCLA Foundation had the highest rate of growth over the previous year, at 49 percent. This article presents a table of the largest college endowments in 2011. The table covers the "rank," "institution," "market value as of June 30, 2011," and "1-year change" of institutions participating in…

  2. Rescue of Infectious Birnavirus from Recombinant Ribonucleoprotein Complexes

    PubMed Central

    Dalton, Romy M.; Rodríguez, José F.

    2014-01-01

    Birnaviruses are unconventional members of the icosahedral double-stranded (dsRNA) RNA virus group. The main differential birnavirus trait is the lack of the inner icosahedral transcriptional core, a ubiquitous structure conserved in all other icosahedral dsRNA viruses, that shelters the genome from cellular dsRNA sensors and provide the enzymatic machinery to produce and extrude mature messenger RNAs. In contrast, birnaviral particles enclose ribonucleoprotein (RNP) complexes formed by the genome segments, the dsRNA-binding VP3 polypeptide and the virus-encoded RNA polymerase (RdRp). The presence of RNPs suggests that the birnavirus replication program might exhibit significant differences with respect to those of prototypal dsRNA viruses. However, experimental evidences supporting this hypothesis are as yet scarce. Of particular relevance for the understanding of birnavirus replication is to determine whether RNPs act as intracellular capsid-independent transcriptional units. Our study was focused to answer this question using the infectious bursal disease virus (IBDV), the best characterized birnavirus, as model virus. Here, we describe the intracellular assembly of functional IBDV RNPs in the absence of the virus-encoded VP2 capsid polypeptide. Recombinant RNPs are generated upon coexpression of the IBDV VP1 and RdRp polypeptides and transfection of purified virus dsRNA. Presented data show that recombinant RNPs direct the expression of the IBDV polypeptide repertoire and the production of infectious virus in culture cells. Results described in this report constitute the first direct experimental evidence showing that birnaviral RNPs are intracellularly active in the absence of the virus capsid. This finding is consistent with presented data indicating that RNP formation precedes virus assembly in IBDV-infected cells, and supports the recently proposed IBDV replication model entailing the release of RNPs during the initial stages of the infection. Indeed

  3. In vitro assembly of a pre-messenger ribonucleoprotein.

    PubMed Central

    Economidis, I V; Pederson, T

    1983-01-01

    Transcription of the Bal I E restriction fragment of adenovirus DNA by RNA polymerase II in a HeLa cell extract produces a RNA transcript 1,712 nucleotides in length. This transcript contains the first two elements of the tripartite leader that, in vivo, is spliced onto the late mRNAs. We have found that this adenovirus 2 transcript forms a specific ribonucleoprotein complex (RNP) in this in vitro system. The RNP particle sediments in sucrose gradients as a monodisperse peak at 50 S and has a buoyant density of 1.34 g/cm3 in Cs2SO4, indicating the same 4:1 protein/RNA composition as native nuclear RNPs that contain pre-mRNA sequences (hnRNP). Moreover, the in vitro-assembled RNP is resistant to concentrations of NaCl that are known to dissociate nonspecific RNA-protein complexes. The adenovirus 2 transcript is precipitated by a monoclonal antibody for hnRNP core proteins. In addition, RNA-protein crosslinking of [alpha-32P]UTP-labeled transcript/RNP complexes reveals that the major proteins in contact with the RNA are the Mr 32,500-41,500 species known to be associated with hnRNA in vivo. These results demonstrate the in vitro assembly of a specific RNA polymerase II transcript into RNP. Moreover, because the 1,712-nucleotide adenovirus 2 transcript lacks poly(A) addition sites and because the leader sequences are not spliced appreciably in this in vitro system, it follows that RNP formation requires neither polyadenylylation nor splicing, nor is it sufficient to cause the latter. Images PMID:6308613

  4. The elemental composition and origin of fine ambient particles in the largest Polish conurbation: first results from the short-term winter campaign

    NASA Astrophysics Data System (ADS)

    Majewski, Grzegorz; Rogula-Kozłowska, Wioletta

    2016-07-01

    Diurnal (24-h) samples of fine particulate matter (PM2.5, ambient particles with an aerodynamic diameter not greater than 2.5 μm) and soil samples were collected in an urban area in Warsaw, in winter. The samples were analysed for 24 elements with an Epsilon 5 spectrometer (PANalytical). The results were then arranged and compared with the results of research conducted earlier in Poland and other parts of the world. Afterwards, sources of ambient PM2.5 were identified and the share of each in the concentration of PM2.5 was evaluated by means of enrichment factor (EF) analysis, principal component analysis (PCA) and multi-linear regression analysis (MLRA). The results were interpreted using a detailed analysis of correlations between diurnal concentrations of PM2.5, PM2.5-elements, and of changes in meteorological conditions. The winter average ambient concentration of PM2.5 in Warsaw, was 10.7 ± 7.5 μg/m3 and was much lower than in the other sites in Poland. In Warsaw, regardless of the concentration of PM2.5, the concentrations of certain PM2.5-bound elements, mainly toxic, were high, e.g. the average ambient concentrations of PM2.5-bound Se, As, Co, V, Cd and Ni were 12.7 ± 30.5, 10.6 ± 34.4, 9.4 ± 13.7, 15.1 ± 32.7, 9.6 ± 22.2 and 3.5 ± 5.0 ng/m3, respectively. The elemental composition and concentrations of PM2.5 appeared to be influenced mainly by the anthropogenic emissions (energy production based on coal and biomass combustion, whose mean contribution to the concentration of PM2.5 was 18.4 %, and energy production based on oil combustion with a contribution of 9.9 % in PM2.5). A mixture of soil matter and road dust was also identified in PM2.5 (8 %). The mean contribution of traffic (exhaust) emissions to the concentration of PM2.5 in an urban area, selected as representative of the Warsaw conurbation, was assessed at 15.4 %.

  5. The elemental composition and origin of fine ambient particles in the largest Polish conurbation: first results from the short-term winter campaign

    NASA Astrophysics Data System (ADS)

    Majewski, Grzegorz; Rogula-Kozłowska, Wioletta

    2015-05-01

    Diurnal (24-h) samples of fine particulate matter (PM2.5, ambient particles with an aerodynamic diameter not greater than 2.5 μm) and soil samples were collected in an urban area in Warsaw, in winter. The samples were analysed for 24 elements with an Epsilon 5 spectrometer (PANalytical). The results were then arranged and compared with the results of research conducted earlier in Poland and other parts of the world. Afterwards, sources of ambient PM2.5 were identified and the share of each in the concentration of PM2.5 was evaluated by means of enrichment factor (EF) analysis, principal component analysis (PCA) and multi-linear regression analysis (MLRA). The results were interpreted using a detailed analysis of correlations between diurnal concentrations of PM2.5, PM2.5-elements, and of changes in meteorological conditions. The winter average ambient concentration of PM2.5 in Warsaw, was 10.7 ± 7.5 μg/m3 and was much lower than in the other sites in Poland. In Warsaw, regardless of the concentration of PM2.5, the concentrations of certain PM2.5-bound elements, mainly toxic, were high, e.g. the average ambient concentrations of PM2.5-bound Se, As, Co, V, Cd and Ni were 12.7 ± 30.5, 10.6 ± 34.4, 9.4 ± 13.7, 15.1 ± 32.7, 9.6 ± 22.2 and 3.5 ± 5.0 ng/m3, respectively. The elemental composition and concentrations of PM2.5 appeared to be influenced mainly by the anthropogenic emissions (energy production based on coal and biomass combustion, whose mean contribution to the concentration of PM2.5 was 18.4 %, and energy production based on oil combustion with a contribution of 9.9 % in PM2.5). A mixture of soil matter and road dust was also identified in PM2.5 (8 %). The mean contribution of traffic (exhaust) emissions to the concentration of PM2.5 in an urban area, selected as representative of the Warsaw conurbation, was assessed at 15.4 %.

  6. Saturn's largest ring.

    PubMed

    Verbiscer, Anne J; Skrutskie, Michael F; Hamilton, Douglas P

    2009-10-22

    Most planetary rings in the Solar System lie within a few radii of their host body, because at these distances gravitational accelerations inhibit satellite formation. The best known exceptions are Jupiter's gossamer rings and Saturn's E ring, broad sheets of dust that extend outward until they fade from view at five to ten planetary radii. Source satellites continuously supply the dust, which is subsequently lost in collisions or by radial transport. Here we report that Saturn has an enormous ring associated with its outer moon Phoebe, extending from at least 128R(S) to 207R(S) (Saturn's radius R(S) is 60,330 km). The ring's vertical thickness of 40R(S) matches the range of vertical motion of Phoebe along its orbit. Dynamical considerations argue that these ring particles span the Saturnian system from the main rings to the edges of interplanetary space. The ring's normal optical depth of approximately 2 x 10(-8) is comparable to that of Jupiter's faintest gossamer ring, although its particle number density is several hundred times smaller. Repeated impacts on Phoebe, from both interplanetary and circumplanetary particle populations, probably keep the ring populated with material. Ring particles smaller than centimetres in size slowly migrate inward and many of them ultimately strike the dark leading face of Iapetus. PMID:19812546

  7. Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex.

    PubMed Central

    Bachmann, M; Mayet, W J; Schröder, H C; Pfeifer, K; Meyer zum Büschenfelde, K H; Müller, W E

    1987-01-01

    45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs. Images Fig. 1. Fig. 3. PMID:2440423

  8. Data on structural transitions in domains of hordeivirus TGB1 protein forming ribonucleoprotein complex.

    PubMed

    Makarov, Valentin V; Makarova, Svetlana S; Kalinina, Natalia O

    2016-09-01

    This data article is related to the research article entitled "in vitro properties of hordeivirus TGB1 protein forming ribonucleoprotein complexes" (Makarov et al., 2015 [1]), demonstrating that upon incubation with viral RNA the poa semilatent hordeivirus (PSLV) TGB1 protein (the movement 63 K protein encoded by the first gene of the triple gene block) in vitro forms RNP structures resembling filamentous virus-like particles and its internal domain (ID) performs a major structural role in this process. This article reports the additional results on the structural lability of ID and the structural transitions in the C-terminal NTPase/helicase domain (HELD) induced by interaction with tRNA and phosphorylation. PMID:27331098

  9. Mechanical Stability and Reversible Fracture of Vault Particles

    PubMed Central

    Llauró, Aida; Guerra, Pablo; Irigoyen, Nerea; Rodríguez, José F.; Verdaguer, Núria; de Pablo, Pedro J.

    2014-01-01

    Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as vehicles for drug delivery. In this article, we examine the local stiffness of individual vaults and probe their structural stability with atomic force microscopy under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we induce single-protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution atomic force microscopy topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which enables these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers. PMID:24507609

  10. Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

    PubMed Central

    Bosser, R; Faura, M; Serratosa, J; Renau-Piqueras, J; Pruschy, M; Bachs, O

    1995-01-01

    It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei. PMID:7823935

  11. Host Factors That Interact with the Pestivirus N-Terminal Protease, Npro, Are Components of the Ribonucleoprotein Complex

    PubMed Central

    Jefferson, Matthew; Donaszi-Ivanov, Andras; Pollen, Sean; Dalmay, Tamas; Saalbach, Gerhard

    2014-01-01

    ABSTRACT The viral N-terminal protease Npro of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for Npro through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to Npro did not inhibit these proteins from aggregating into stress granules. Npro interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with Npro. To address a proviral role for Npro in RNP granules, we investigated whether Npro affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of Npro had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that Npro is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. IMPORTANCE Although the pestivirus N-terminal protease, Npro, has been shown to have an important role in degrading IRF3 to

  12. Largest Solar Flare on Record

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The largest solar flare ever recorded occurred at 4:51 p.m. EDT, on Monday, April 2, 2001. as Observed by the Solar and Heliospheric Observatory (SOHO) satellite. Solar flares, among the solar systems mightiest eruptions, are tremendous explosions in the atmosphere of the Sun capable of releasing as much energy as a billion megatons of TNT. Caused by the sudden release of magnetic energy, in just a few seconds, solar flares can accelerate solar particles to very high velocities, almost to the speed of light, and heat solar material to tens of millions of degrees. The recent explosion from the active region near the sun's northwest limb hurled a coronal mass ejection into space at a whopping speed of roughly 7.2 million kilometers per hour. Luckily, the flare was not aimed directly towards Earth. Second to the most severe R5 classification of radio blackout, this flare produced an R4 blackout as rated by the NOAA SEC. This classification measures the disruption in radio communications. Launched December 2, 1995 atop an ATLAS-IIAS expendable launch vehicle, the SOHO is a cooperative effort involving NASA and the European Space Agency (ESA). (Image courtesy NASA Goddard SOHO Project office)

  13. Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

    PubMed Central

    Wang, Chen; Meier, U Thomas

    2004-01-01

    Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57–NOP10–NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein–RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis. PMID:15044956

  14. Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo.

    PubMed Central

    Economidis, I V; Pederson, T

    1983-01-01

    Mouse erythroleukemia cells were pulse-labeled with [3H]uridine and irradiated with 254-nm light to produce covalent crosslinks between RNA and proteins in close proximity to one another in vivo. Nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA were isolated and digested with nucleases, and the resulting proteins were subjected to gel electrophoresis. Proteins carrying covalently crosslinked [3H]uridine nucleotides were identified by fluorography. The results demonstrate that heterogeneous nuclear RNA is complexed in vivo with a set of six major proteins having molecular weights between 32,500 and 41,500. Analysis of chromatin fractions indicates that nascent heterogeneous nuclear RNA chains assemble with these six proteins as a very early post-transcriptional event. These data, and other results [Nevins, J. R. & Darnell, J. E. (1981) Cell 15, 1477-1493], lead us to propose the usual order of post-transcriptional events to be: heterogeneous nuclear RNA-ribonucleoprotein particle assembly leads to poly(A) addition leads to splicing. Images PMID:6572923

  15. RNA–protein binding interface in the telomerase ribonucleoprotein

    PubMed Central

    Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad R.; Nelson, Randall W.; Chen, Julian J.-L.

    2011-01-01

    Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR–TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA–protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain. PMID:22123986

  16. Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA

    SciTech Connect

    Liang, Bo; Zhou, Jing; Kahen, Elliot; Terns, Rebecca M.; Terns, Michael P.; Li, Hong

    2009-09-29

    Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 {angstrom} of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

  17. The structure and function of small nucleolar ribonucleoproteins

    PubMed Central

    Reichow, Steve L.; Hamma, Tomoko; Ferré-D'Amaré, Adrian R.; Varani, Gabriele

    2007-01-01

    Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequence-specific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein–RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose-2′-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the core archaeal sRNP proteins have been determined and their assembly pathways delineated. Furthermore, the recent structure of an H/ACA complex has revealed the organization of a complete sRNP. Combined with current biochemical data, these structures offer insight into the highly homologous eukaryotic snoRNPs. PMID:17284456

  18. High-Throughput Analysis of RNA Structure and Ribonucleoprotein Assembly

    PubMed Central

    McGinnis, Jennifer L.; Duncan, Caia D. S.; Weeks, Kevin M.

    2016-01-01

    RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and non-additive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes. PMID:20946765

  19. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    PubMed

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. PMID:27130213

  20. Structural stability of vault particles.

    PubMed

    Esfandiary, Reza; Kickhoefer, Valerie A; Rome, Leonard H; Joshi, Sangeeta B; Middaugh, C Russell

    2009-04-01

    Vaults, at 13 MDa, are the largest ribonucleoprotein particles known. In vitro, expression of the major vault protein (MVP) alone in Sf9 insect cells results in the production of recombinant particles with characteristic vault structure. With the ultimate goal of using recombinant vaults as nanocapsules for the delivery of biomolecules, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy, and light scattering) along with electron microscopy, to characterize the structural stability of vaults over a wide range of pH (3-8) and temperature (10-90 degrees C). Ten different conformational states of the vaults were identified over the pH and temperature range studied with the most stable region at pH 6-8 below 40 degrees C and least stable at pH 4-6 above 60 degrees C. A unique intermediate molten globulelike state was also identified at pH 6 and approximately 55 degrees C. EM imaging showed the opening of intact vaults into flowerlike structures when transitioning from neutral to acidic pH. This information has potential use in the development of recombinant vaults into nanocapsules for drug delivery since one mechanism by which therapeutic agents entrapped in vaults could be released is through an opening of the intact vault structure. PMID:18683860

  1. Largest impact craters on Venus

    NASA Technical Reports Server (NTRS)

    Ivanov, B. A.; Weitz, C. M.; Basilevsky, A. T.

    1992-01-01

    High-resolution radar images from the Magellan spacecraft have allowed us to perform a detailed study on 25 large impact craters on Venus with diameters from 70 to 280 km. The dimension of these large craters is comparable with the characteristic thickness of the venusian lithosphere and the atmospheric scale height. Some physical parameters for the largest impact craters on Venus (LICV), such as depth, ring/diameter ratio, and range of ballistic ejecta deposits, have been obtained from the SAR images and the altimetry dataset produced by MIT. Data related to each of these parameters is discussed.

  2. The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes

    PubMed Central

    Turrell, Lauren; Lyall, Jon W.; Tiley, Laurence S.; Fodor, Ervin; Vreede, Frank T.

    2014-01-01

    The nucleoprotein of negative strand RNA viruses forms a major component of the ribonucleoprotein complex that is responsible for viral transcription and replication. However, the precise role of nucleoprotein in viral RNA transcription and replication is not clear. Here we show that nucleoprotein of influenza A virus is entirely dispensable for replication and transcription of short viral RNA-like templates in vivo, suggesting that nucleoprotein represents an elongation factor for the viral RNA polymerase. We also find that the recruitment of nucleoprotein to nascent ribonucleoprotein complexes during replication of full length viral genes is mediated through nucleoprotein-nucleoprotein homo-oligomerisation in a “tail loop-first” orientation and is independent of RNA binding. This work demonstrates that nucleoprotein does not regulate the initiation and termination of transcription and replication by the viral polymerase in vivo and provides new mechanistic insights into the assembly and regulation of viral ribonucleoprotein complexes. PMID:23481399

  3. Rehabilitating China's largest inland river.

    PubMed

    Li, Yiqing; Chen, Yaning; Zhang, Yaoqi; Xia, Yang

    2009-06-01

    Wetlands are particularly important for conserving China's biodiversity but riparian wetlands in the Tarim River basin in western China have been reduced by 46% during the last 3 decades. The world's largest habitat for Populus euphratica, which is in the Tarim River basin, significantly shrank. To protect and restore the deteriorated ecosystems along the Tarim River and its associated wetlands, China's government initiated a multimillion dollar river restoration project to release water from upper dams to the dried-up lower reaches of the Tarim River starting in 2000. We monitored the responses of groundwater and vegetation to water recharge in the lower reaches of the river from 2000 to 2006 by establishing nine 1000-m-long transects perpendicular to the river at intervals of 20-45 km along the 320-km river course below the Daxihaizi Reservoir, the source of water conveyance, to Lake Taitema, the terminus of the Tarim River. Water recharges from the Daxihaizi Reservoir to the lower reaches of the Tarim River significantly increased groundwater levels and vegetation coverage at all monitoring sites along the river. The mean canopy size of the endangered plant species P. euphratica doubled after 6 years of water recharge. Some rare migrating birds returned to rest on the restored wetlands in summer along the lower reaches of the Tarim River. The biggest challenge facing decision makers, however, is to balance water allocation and water rights between agricultural and natural ecosystems in a sustainable way. A large number of inhabitants in the Tarim Basin depend on these limited water resources for a living. At the same time, the endangered ecosystems need to be protected. Given the ecological, socioeconomic, and sociopolitical realities in the Tarim Basin, adaptive water policies and strategies are needed for water allocation in these areas of limited water resources. PMID:22748091

  4. The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

    PubMed Central

    Chapman, K S; Ingle, J

    1976-01-01

    A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA. PMID:1008819

  5. A Seasonal Trend of Single Scattering Albedo in Southern African Biomass-burning Particles: Implications for Satellite Products and Estimates of Emissions for the World's Largest Biomass-burning Source

    NASA Technical Reports Server (NTRS)

    Eck, T. F.; Holben, B. N.; Reid, J. S.; Mukelabai, M. M.; Piketh, S. J.; Torres, O.; Jethva, H. T.; Hyer, E. J.; Ward, D. E.; Dubovik, O.; Sinyuk, A.; Schafer, J. S.; Giles, D. M.; Sorokin, M.; Smirnov, A.; Slutsker, I.

    2013-01-01

    As a representative site of the southern African biomass-burning region, sun-sky data from the 15 year Aerosol Robotic Network (AERONET) deployment at Mongu, Zambia, was analyzed. For the biomass-burning season months (July-November), we investigate seasonal trends in aerosol single scattering albedo (SSA), aerosol size distributions, and refractive indices from almucantar sky scan retrievals. The monthly mean single scattering albedo at 440 nm in Mongu was found to increase significantly from approx.. 0.84 in July to approx. 0.93 in November (from 0.78 to 0.90 at 675 nm in these same months). There was no significant change in particle size, in either the dominant accumulation or secondary coarse modes during these months, nor any significant trend in the Angstrom exponent (440-870 nm; r(exp 2) = 0.02). A significant downward seasonal trend in imaginary refractive index (r(exp 2) = 0.43) suggests a trend of decreasing black carbon content in the aerosol composition as the burning season progresses. Similarly, burning season SSA retrievals for the Etosha Pan, Namibia AERONET site also show very similar increasing single scattering albedo values and decreasing imaginary refractive index as the season progresses. Furthermore, retrievals of SSA at 388 nm from the Ozone Monitoring Instrument satellite sensor show similar seasonal trends as observed by AERONET and suggest that this seasonal shift is widespread throughout much of southern Africa. A seasonal shift in the satellite retrieval bias of aerosol optical depth from the Moderate Resolution Imaging Spectroradiometer collection 5 dark target algorithm is consistent with this seasonal SSA trend since the algorithm assumes a constant value of SSA. Multi-angle Imaging Spectroradiometer, however, appears less sensitive to the absorption-induced bias.

  6. Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N

    SciTech Connect

    McAllister, G.; Amara, S.G.; Lerner, M.R. )

    1988-07-01

    Sera from some patients with systemic lupus erythematosus and other autoimmune diseases have antibodies against nuclear antigens. An example is anti-Sm sera, which recognize proteins associated with small nuclear RNA molecules (small nuclear ribonucleoprotein (snRNP) particles). In this paper anti-Sm sera were used to probe immunoblots of various rat tissues. A previously unidentified M{sub r} 28,000 polypeptide was recognized by these anti-Sm sera. This polypeptide, referred to as N, is expressed in a tissue-specific manner, being most abundant in rat brain, less so in heart, and undetectable in the other tissues examined. Immunoprecipitation experiments using antibodies directed against the cap structure of small nuclear RNAs have demonstrated that N is a snRNP-associated polypeptide. Anti-Sm serum was also used to isolate a partial cDNA clone ({lambda}rb91) from a rat brain phage {lambda}gt11 cDNA expression library. A longer cDNA clone was obtained by rescreening the library with {lambda}rb91. In vitro transcription and subsequent translation of this subcloned, longer insert (pGMA2) resulted in a protein product with the same electrophoretic and immunological properties as N, confirming that pGMA2 encodes N. The tissue distribution of N and the involvement of snRNP particles in nuclear pre-mRNA processing may imply a role for N in tissue-specific pre-mRNA splicing.

  7. Conserved residues in Lassa fever virus Z protein modulate viral infectivity at the level of the ribonucleoprotein.

    PubMed

    Capul, Althea A; de la Torre, Juan Carlos; Buchmeier, Michael J

    2011-04-01

    Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles. PMID:21228230

  8. Disassembly of Human Immunodeficiency Virus Type 1 Cores In Vitro Reveals Association of Nef with the Subviral Ribonucleoprotein Complex

    PubMed Central

    Forshey, Brett M.; Aiken, Christopher

    2003-01-01

    The human immunodeficiency virus type 1 (HIV-1) virulence factor Nef enhances viral infectivity in single-cycle infection assays and accelerates HIV-1 replication in vitro. It has been reported that the effects of Nef are mediated early after viral entry and before the completion of reverse transcription, as viral DNA synthesis is strongly attenuated during infection by Nef-defective virions. Our previous work has demonstrated that Nef is associated with mature HIV-1 cores, implicating Nef in the regulation of HIV-1 core stability. Here we report a comparative analysis of HIV-1 cores isolated from wild-type and Nef-defective particles. We observed no effect of Nef on HIV-1 core structure or stability; however, Nef cosedimented with a subviral ribonucleoprotein complex following dissociation of CA. These results indicate that Nef interacts tightly with an internal component of the HIV-1 core. They further suggest that virion-associated Nef may facilitate an early step in HIV-1 infection following dissociation of the viral capsid in the target cell. PMID:12634398

  9. The poly(adenylic acid)-protein complex is restricted to the nonpolysomal messenger ribonucleoprotein of Physarum polycephalum.

    PubMed

    Adams, D S; Noonan, D; Jeffery, W R

    1980-04-29

    The distribution of poly(adenylic acid) [poly(A)]-protein complexes in the polysomal and nonpolysomal messenger ribonucleoprotein (mRNP) fractions of Physarum polycephalum was examined in the present study. Poly-(A)-containing components released from the nonpolysomal mRNP by ribonuclease (RNase) digestion were quantitatively adsorbed to nitrocellulose filters at low ionic strength, were highly resistant to micrococcal nuclease under conditions in which free poly(A) was completely degraded, and sedimented as a 10-15S particle which was disrupted by sodium dodecyl sulfate and protease treatment. These are characteristics of the poly(A)-protein complex. In contrast,poly(A)-containing molecules released from the polysomes by RNase were refractive to nitrocellulose, were completely sensitive to micrococcal nuclease, and sedimented at 2-4 S, identical with the sedimentation exhibited by protein-free poly(A). Examination of the poly(A) sequences present in polysomal and nonpolysomal mRNP by polyacylamide gel electrophoresis showed that the former contained only very short sequences, averaging approximately 15 nucleotides, while the latter exhibited only much longer segments, averaging approximately 65 nucleotides. It is concluded that poly(A)-protein complexes are restricted to the nonpolysomal mRNP of Physarum and that the limiting factor in complex formation may be the length of the available poly(A) binding site. PMID:7378386

  10. Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

    PubMed

    Singh, Deepali; Boeras, Ioana; Singh, Gatikrushna; Boris-Lawrie, Kathleen

    2016-01-01

    All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification. PMID:26714709

  11. Autoantibody to Th ribonucleoprotein (nucleolar 7-2 RNA protein particle) in patients with systemic sclerosis

    SciTech Connect

    Okano, Y.; Medsger, T.A. Jr. )

    1990-12-01

    We studied sera of 371 consecutive new patients with systemic sclerosis (SSc; scleroderma) who were first evaluated during 1984-1988. All sera were tested for antinuclear antibodies by immunofluorescence staining using HEp-2 cells as substrate. We excluded 219 sera showing dark nucleoli and screened for antibodies to Th in the remaining 152 sera by immunoprecipitation of a 32P-labeled HeLa cell extract. Fifteen (4.0%) of 371 sera were anti-Th+. Anti-Th antibodies were present in 14 (8.4%) of 167 SSc patients with limited cutaneous involvement, in 1 of 167 with diffuse cutaneous involvement, and in 0 of 37 with SSc overlap syndrome. Among 244 controls with other connective tissue diseases, anti-Th was detected in only 3 patients, all having primary Raynaud's phenomenon of less than 2 years duration. In the subgroup with SSc with limited cutaneous involvement, the 14 anti-Th+ patients had a significantly greater frequency of puffy fingers, small bowel involvement, and hypothyroidism, and a significantly lower frequency of arthralgia and/or arthritis. Their cumulative survival rate from the time of onset of symptoms was lower than that for anti-Th- patients (78% versus 91% at 10 years), primarily due to 3 deaths from pulmonary arterial hypertension (2 from primary pulmonary hypertension and 1 from pulmonary hypertension secondary to pulmonary interstitial fibrosis). Serum anti-Th antibodies are present almost exclusively in patients with SSc with limited cutaneous involvement or in those with primary Raynaud's phenomenon whose disease may evolve to SSc with limited cutaneous involvement, and these antibodies may identify those patients who are at greater risk for reduced survival.

  12. Purification and visualization of influenza a viral ribonucleoprotein complexes.

    PubMed

    Wu, Winco W H; Weaver, Lindsay L; Panté, Nelly

    2009-01-01

    The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)(1), and the negative staining has been performed by Wu et al. (2007).(2). PMID:19229188

  13. An improved version of the hairpin ribozyme functions as a ribonucleoprotein complex.

    PubMed

    Sargueil, B; Pecchia, D B; Burke, J M

    1995-06-13

    Most RNA molecules that are endowed with catalytic activity function in the form of ribonucleoproteins within cells. These complexes are frequently large, poorly defined, and difficult to study. As a model system to study biological catalysis by ribonucleoproteins, we have modified the hairpin ribozyme by inserting an RNA structure that serves as a binding site for bacteriophage R17 coat protein in the form of an extension to ribozyme helix 4, which lies at the periphery of the catalytic domain. In the absence of protein, we find that incorporation of the protein-binding domain increases the catalytic efficiency of the hairpin ribozyme by 2-fold for the cleavage reaction and 16-fold for the ligation reaction. This increase in activity correlates with an increase in the proportion of molecules which fold into the active tertiary structure, as measured by a UV cross-linking assay. Mobility-shift and filter-binding assays of complex formation show that R17 coat protein binds to the chimeric ribozyme with a dissociation constant essentially identical to that of the isolated protein-binding domain; no binding of the protein to the unmodified ribozyme could be detected. The kinetics of cleavage and ligation reactions are not altered by the presence of saturating concentrations of coat protein, and competition studies demonstrate that the protein remains bound to the ribozyme throughout the catalytic cycle. These studies establish that the hairpin ribozyme can be engineered to function efficiently in the form of a ribonucleoprotein in vitro and will serve as the basis for future experimentation to understand mechanisms of protein modulation of catalytic RNA activity, and to introduce other protein-binding domains, for example, HIV-1 rev-binding and tar elements, which may be useful for influencing subcellular localization, regulating intracellular activity, or generating ribozymes that also function as "decoys" in antiviral applications. PMID:7779820

  14. Rabies Group-Specific Ribonucleoprotein Antigen and a Test System for Grouping and Typing of Rhabdoviruses

    PubMed Central

    Schneider, L. G.; Dietzschold, B.; Dierks, R. E.; Matthaeus, W.; Enzmann, P.-J.; Strohmaier, K.

    1973-01-01

    Cell-associated ribonucleoprotein (RNP) was isolated from BHK-21 cells infected with several strains of rabies and rabies-related viruses. The RNP-antigen from rabies and related viruses induced the formation of complement-fixing, precipitating, and immunofluorescent antibodies, and proved to be the group-specific antigen common to all rabies viruses. Antigens of the envelope which induce virus-neutralizing antibodies are apparently determinative for the serotype of a virus as evidenced by two-way neutralization tests. A combination of these methods seems to be a useful approach to the serological grouping and typing of rhabdoviruses. Images PMID:4196634

  15. Assembly of vault-like particles in insect cells expressing only the major vault protein.

    PubMed

    Stephen, A G; Raval-Fernandes, S; Huynh, T; Torres, M; Kickhoefer, V A; Rome, L H

    2001-06-29

    Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes approximately 75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo. PMID:11349122

  16. Germany gets largest ever MRI scanner

    NASA Astrophysics Data System (ADS)

    Stafford, Ned

    2008-07-01

    A 57-tonne cylindrical magnet has arrived at the Jülich Research Centre in Germany, where physicists are putting together the world's largest magnetic resonance imaging (MRI) system. When the machine comes online next year, medical researchers will use it to examine the brain in unprecedented detail, in the hope of gaining new insights into neurodegenerative diseases such as Alzheimer's.

  17. Air pollution ranks as largest health risk

    NASA Astrophysics Data System (ADS)

    Wendel, JoAnna

    2014-04-01

    The World Health Organization (WHO) reports that 7 million people died in 2012 from air-pollution-related sicknesses, marking air pollution as the single largest environmental health risk. This finding, a result of better knowledge and assessment of the diseases, is more than double previous estimates of the risk of death from air pollution.

  18. Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components*

    PubMed Central

    Jacobs, Jessica; Marx, Christina; Kock, Vera; Reifschneider, Olga; Fränzel, Benjamin; Krisp, Christoph; Wolters, Dirk; Kück, Ulrich

    2013-01-01

    Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns. PMID:23559604

  19. Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins.

    PubMed Central

    Srisawat, C; Engelke, D R

    2001-01-01

    RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs. PMID:11345441

  20. Saga is largest commercial submarine ever

    SciTech Connect

    Not Available

    1985-05-01

    The long-range autonomous submarine, Saga, went nuclear last year with an agreement between the French and two Canadian companies. The agreement to convert the prototype from Swedish Stirling closed-cycle combustion engines to a nuclear power supply will make Saga the first non-defense nuclear submarine. With an external hull displacement of 500 tons, Saga will be the largest commercial submarine ever built.

  1. Flight performance of the largest volant bird

    PubMed Central

    Ksepka, Daniel T.

    2014-01-01

    Pelagornithidae is an extinct clade of birds characterized by bizarre tooth-like bony projections of the jaws. Here, the flight capabilities of pelagornithids are explored based on data from a species with the largest reported wingspan among birds. Pelagornis sandersi sp. nov. is represented by a skull and substantial postcranial material. Conservative wingspan estimates (∼6.4 m) exceed theoretical maximums based on extant soaring birds. Modeled flight properties indicate that lift:drag ratios and glide ratios for P. sandersi were near the upper limit observed in extant birds and suggest that pelagornithids were highly efficient gliders, exploiting a long-range soaring ecology. PMID:25002475

  2. Fiji's largest marine reserve benefits reef sharks

    NASA Astrophysics Data System (ADS)

    Goetze, J. S.; Fullwood, L. A. F.

    2013-03-01

    To provide more information about whether sharks benefit from no-take marine reserves, we quantified the relative abundance and biomass of reef sharks inside and outside of Namena, Fiji's largest reserve (60.6 km2). Using stereo baited remote underwater video systems (stereo-BRUVs), we found that the abundance and biomass of sharks was approximately two and four times greater in shallow and deep locations, respectively, within the Namena reserve compared to adjacent fished areas. The greater abundance and biomass of reef sharks inside Namena is likely a result of greater prey availability rather than protection from fishing. This study demonstrates that marine reserves can benefit sharks.

  3. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

    SciTech Connect

    Loucaides, Eva M.; Kirchbach, Johann C. von; Foeglein, Agnes; Sharps, Jane; Fodor, Ervin; Digard, Paul

    2009-11-10

    The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

  4. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

    PubMed Central

    Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

    2007-01-01

    Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

  5. catRAPID signature: identification of ribonucleoproteins and RNA-binding regions

    PubMed Central

    Livi, Carmen Maria; Klus, Petr; Delli Ponti, Riccardo; Tartaglia, Gian Gaetano

    2016-01-01

    Motivation: Recent technological advances revealed that an unexpected large number of proteins interact with transcripts even if the RNA-binding domains are not annotated. We introduce catRAPID signature to identify ribonucleoproteins based on physico-chemical features instead of sequence similarity searches. The algorithm, trained on human proteins and tested on model organisms, calculates the overall RNA-binding propensity followed by the prediction of RNA-binding regions. catRAPID signature outperforms other algorithms in the identification of RNA-binding proteins and detection of non-classical RNA-binding regions. Results are visualized on a webpage and can be downloaded or forwarded to catRAPID omics for predictions of RNA targets. Availability and implementation: catRAPID signature can be accessed at http://s.tartaglialab.com/new_submission/signature. Contact: gian.tartaglia@crg.es or gian@tartaglialab.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26520853

  6. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

    PubMed Central

    Loucaides, Eva M.; von Kirchbach, Johann C.; Foeglein, Ágnes; Sharps, Jane; Fodor, Ervin; Digard, Paul

    2009-01-01

    The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity. PMID:19744689

  7. tRNA Core Hypothesis for the Transition from the RNA World to the Ribonucleoprotein World

    PubMed Central

    de Farias, Savio T.; Rêgo, Thais G.; José, Marco V.

    2016-01-01

    Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA) and the peptidyl transferase center (rRNA), proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA. PMID:27023615

  8. tRNA Core Hypothesis for the Transition from the RNA World to the Ribonucleoprotein World.

    PubMed

    de Farias, Savio T; Rêgo, Thais G; José, Marco V

    2016-01-01

    Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA) and the peptidyl transferase center (rRNA), proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA. PMID:27023615

  9. A structurally plastic ribonucleoprotein complex mediates post-transcriptional gene regulation in HIV-1.

    PubMed

    Fernandes, Jason D; Booth, David S; Frankel, Alan D

    2016-07-01

    HIV replication requires the nuclear export of essential, intron-containing viral RNAs. To facilitate export, HIV encodes the viral accessory protein Rev which binds unspliced and partially spliced viral RNAs and creates a ribonucleoprotein complex that recruits the cellular Chromosome maintenance factor 1 export machinery. Exporting RNAs in this manner bypasses the necessity for complete splicing as a prerequisite for mRNA export, and allows intron-containing RNAs to reach the cytoplasm intact for translation and virus packaging. Recent structural studies have revealed that this entire complex exhibits remarkable plasticity at many levels of organization, including RNA folding, protein-RNA recognition, multimer formation, and host factor recruitment. In this review, we explore each aspect of plasticity from structural, functional, and possible therapeutic viewpoints. WIREs RNA 2016, 7:470-486. doi: 10.1002/wrna.1342 For further resources related to this article, please visit the WIREs website. PMID:26929078

  10. Cytoplasmic Accumulation of Heterogeneous Nuclear Ribonucleoprotein K Strongly Promotes Tumor Invasion in Renal Cell Carcinoma Cells

    PubMed Central

    Otoshi, Taiyo; Tanaka, Tomoaki; Morimoto, Kazuya; Nakatani, Tatsuya

    2015-01-01

    Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a part of the ribonucleoprotein complex which regulates diverse biological events. While overexpression of hnRNP K has been shown to be related to tumorigenesis in several cancers, both the expression patterns and biological mechanisms of hnRNP K in renal cell carcinoma (RCC) cells remain unclear. In this study, we showed that hnRNP K protein was strongly expressed in selected RCC cell lines (ACHN, A498, Caki-1, 786–0), and knock-down of hnRNP K expression by siRNA induced cell growth inhibition and apoptosis. Based on immunohistochemical (IHC) analysis of hnRNP K expression in human clear cell RCC specimens, we demonstrated that there was a significant positive correlation between hnRNP K staining score and tumor aggressiveness (e.g., Fuhrman grade, metastasis). Particularly, the rate of cytoplasmic localization of hnRNP K in primary RCC with distant metastasis was significantly higher than that in RCC without metastasis. Additionally, our results indicated that the cytoplasmic distribution of hnRNP K induced by TGF-β stimulus mainly contributed to TGF-β-triggered tumor cell invasion in RCC cells. Dominant cytoplasmic expression of ectopic hnRNP K markedly suppressed the inhibition of invasion by knock-down of endogenous hnRNP K. The expression level of matrix metalloproteinase protein-2 was decreased by endogenous hnRNP K knock-down, and restored by ectopic hnRNP K. Therefore, hnRNP K may be a key molecule involved in cell motility in RCC cells, and molecular mechanism associated with the subcellular localization of hnRNP K may be a novel target in the treatment of metastatic RCC. PMID:26713736

  11. Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease*

    PubMed Central

    Diner, Ian; Hales, Chadwick M.; Bishof, Isaac; Rabenold, Lake; Duong, Duc M.; Yi, Hong; Laur, Oskar; Gearing, Marla; Troncoso, Juan; Thambisetty, Madhav; Lah, James J.; Levey, Allan I.; Seyfried, Nicholas T.

    2014-01-01

    Recent evidence indicates that U1-70K and other U1 small nuclear ribonucleoproteins are Sarkosyl-insoluble and associate with Tau neurofibrillary tangles selectively in Alzheimer disease (AD). Currently, the mechanisms underlying the conversion of soluble nuclear U1 small nuclear ribonucleoproteins into insoluble cytoplasmic aggregates remain elusive. Based on the biochemical and subcellular distribution properties of U1-70K in AD, we hypothesized that aggregated U1-70K itself or other biopolymers (e.g. proteins or nucleic acids) interact with and sequester natively folded soluble U1-70K into insoluble aggregates. Here, we demonstrate that total homogenates from AD brain induce soluble U1-70K from control brain or recombinant U1-70K to become Sarkosyl-insoluble. This effect was not dependent on RNA and did not correlate with detergent-insoluble Tau levels as AD homogenates with reduced levels of these components were still capable of inducing U1-70K aggregation. In contrast, proteinase K-treated AD homogenates and Sarkosyl-soluble AD fractions were unable to induce U1-70K aggregation, indicating that aggregated proteins in AD brain are responsible for inducing soluble U1-70K aggregation. It was determined that the C terminus of U1-70K, which harbors two disordered low complexity (LC) domains, is necessary for U1-70K aggregation. Moreover, both LC1 and LC2 domains were sufficient for aggregation. Finally, protein cross-linking and mass spectrometry studies demonstrated that a U1-70K fragment harboring the LC1 domain directly interacts with aggregated U1-70K in AD brain. Our results support a hypothesis that aberrant forms of U1-70K in AD can directly sequester soluble forms of U1-70K into insoluble aggregates. PMID:25355317

  12. Diversity of a ribonucleoprotein family in tobacco chloroplasts: two new chloroplast ribonucleoproteins and a phylogenetic tree of ten chloroplast RNA-binding domains.

    PubMed Central

    Ye, L H; Li, Y Q; Fukami-Kobayashi, K; Go, M; Konishi, T; Watanabe, A; Sugiura, M

    1991-01-01

    Two new ribonucleoproteins (RNPs) have been identified from a tobacco chloroplast lysate. These two proteins (cp29A and cp29B) are nuclear-encoded and have a less affinity to single-stranded DNA as compared with three other chloroplast RNPs (cp28, cp31 and cp33) previously isolated. DNA sequencing revealed that both contain two consensus sequence-type homologous RNA-binding domains (CS-RBDs) and a very acidic amino-terminal domain but shorter than that of cp28, cp31 and cp33. Comparison of cp29A and cp29B showed a 19 amino acid insertion in the region separating the two CS-RBDs in cp29B. This insertion results in three tandem repeats of a glycine-rich sequence of 10 amino acids, which is a novel feature in RNPs. The two proteins are encoded by different single nuclear genes and no alternatively spliced transcripts could be identified. We constructed a phylogenetic tree for the ten chloroplast CS-RBDs. These results suggest that there is a sizable RNP family in chloroplasts and the diversity was mainly generated through a series of gene duplications rather than through alternative pre-mRNA splicing. The gene for cp29B contains three introns. The first and second introns interrupt the first CS-RBD and the third intron does the second CS-RBD. The position of the first intron site is the same as that in the human hnRNP A1 protein gene. Images PMID:1721701

  13. Collapse of the world's largest herbivores.

    PubMed

    Ripple, William J; Newsome, Thomas M; Wolf, Christopher; Dirzo, Rodolfo; Everatt, Kristoffer T; Galetti, Mauro; Hayward, Matt W; Kerley, Graham I H; Levi, Taal; Lindsey, Peter A; Macdonald, David W; Malhi, Yadvinder; Painter, Luke E; Sandom, Christopher J; Terborgh, John; Van Valkenburgh, Blaire

    2015-05-01

    Large wild herbivores are crucial to ecosystems and human societies. We highlight the 74 largest terrestrial herbivore species on Earth (body mass ≥100 kg), the threats they face, their important and often overlooked ecosystem effects, and the conservation efforts needed to save them and their predators from extinction. Large herbivores are generally facing dramatic population declines and range contractions, such that ~60% are threatened with extinction. Nearly all threatened species are in developing countries, where major threats include hunting, land-use change, and resource depression by livestock. Loss of large herbivores can have cascading effects on other species including large carnivores, scavengers, mesoherbivores, small mammals, and ecological processes involving vegetation, hydrology, nutrient cycling, and fire regimes. The rate of large herbivore decline suggests that ever-larger swaths of the world will soon lack many of the vital ecological services these animals provide, resulting in enormous ecological and social costs. PMID:26601172

  14. Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection.

    PubMed

    Newkirk, M M; van Venrooij, W J; Marshall, G S

    2001-01-01

    The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus. PMID:11438044

  15. The largest volcanic eruptions on Earth

    NASA Astrophysics Data System (ADS)

    Bryan, Scott E.; Peate, Ingrid Ukstins; Peate, David W.; Self, Stephen; Jerram, Dougal A.; Mawby, Michael R.; Marsh, J. S. (Goonie); Miller, Jodie A.

    2010-10-01

    Large igneous provinces (LIPs) are sites of the most frequently recurring, largest volume basaltic and silicic eruptions in Earth history. These large-volume (> 1000 km 3 dense rock equivalent) and large-magnitude (> M8) eruptions produce areally extensive (10 4-10 5 km 2) basaltic lava flow fields and silicic ignimbrites that are the main building blocks of LIPs. Available information on the largest eruptive units are primarily from the Columbia River and Deccan provinces for the dimensions of flood basalt eruptions, and the Paraná-Etendeka and Afro-Arabian provinces for the silicic ignimbrite eruptions. In addition, three large-volume (675-2000 km 3) silicic lava flows have also been mapped out in the Proterozoic Gawler Range province (Australia), an interpreted LIP remnant. Magma volumes of > 1000 km 3 have also been emplaced as high-level basaltic and rhyolitic sills in LIPs. The data sets indicate comparable eruption magnitudes between the basaltic and silicic eruptions, but due to considerable volumes residing as co-ignimbrite ash deposits, the current volume constraints for the silicic ignimbrite eruptions may be considerably underestimated. Magma composition thus appears to be no barrier to the volume of magma emitted during an individual eruption. Despite this general similarity in magnitude, flood basaltic and silicic eruptions are very different in terms of eruption style, duration, intensity, vent configuration, and emplacement style. Flood basaltic eruptions are dominantly effusive and Hawaiian-Strombolian in style, with magma discharge rates of ~ 10 6-10 8 kg s -1 and eruption durations estimated at years to tens of years that emplace dominantly compound pahoehoe lava flow fields. Effusive and fissural eruptions have also emplaced some large-volume silicic lavas, but discharge rates are unknown, and may be up to an order of magnitude greater than those of flood basalt lava eruptions for emplacement to be on realistic time scales (< 10 years). Most

  16. The largest volcanic eruptions on Earth

    NASA Astrophysics Data System (ADS)

    Bryan, Scott E.; Peate, Ingrid Ukstins; Peate, David W.; Self, Stephen; Jerram, Dougal A.; Mawby, Michael R.; Marsh, J. S. (Goonie); Miller, Jodie A.

    2010-10-01

    Large igneous provinces (LIPs) are sites of the most frequently recurring, largest volume basaltic and silicic eruptions in Earth history. These large-volume (> 1000 km 3 dense rock equivalent) and large-magnitude (> M8) eruptions produce areally extensive (10 4-10 5 km 2) basaltic lava flow fields and silicic ignimbrites that are the main building blocks of LIPs. Available information on the largest eruptive units are primarily from the Columbia River and Deccan provinces for the dimensions of flood basalt eruptions, and the Paraná-Etendeka and Afro-Arabian provinces for the silicic ignimbrite eruptions. In addition, three large-volume (675-2000 km 3) silicic lava flows have also been mapped out in the Proterozoic Gawler Range province (Australia), an interpreted LIP remnant. Magma volumes of > 1000 km 3 have also been emplaced as high-level basaltic and rhyolitic sills in LIPs. The data sets indicate comparable eruption magnitudes between the basaltic and silicic eruptions, but due to considerable volumes residing as co-ignimbrite ash deposits, the current volume constraints for the silicic ignimbrite eruptions may be considerably underestimated. Magma composition thus appears to be no barrier to the volume of magma emitted during an individual eruption. Despite this general similarity in magnitude, flood basaltic and silicic eruptions are very different in terms of eruption style, duration, intensity, vent configuration, and emplacement style. Flood basaltic eruptions are dominantly effusive and Hawaiian-Strombolian in style, with magma discharge rates of ~ 10 6-10 8 kg s -1 and eruption durations estimated at years to tens of years that emplace dominantly compound pahoehoe lava flow fields. Effusive and fissural eruptions have also emplaced some large-volume silicic lavas, but discharge rates are unknown, and may be up to an order of magnitude greater than those of flood basalt lava eruptions for emplacement to be on realistic time scales (< 10 years). Most

  17. The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes.

    PubMed Central

    Haynes, S R; Raychaudhuri, G; Beyer, A L

    1990-01-01

    The Drosophila Hrb98DE locus encodes proteins that are highly homologous to the mammalian A1 protein, a major component of heterogeneous nuclear ribonucleoprotein (RNP) particles. The Hrb98DE locus is transcribed throughout development, with the highest transcript levels found in ovaries, early embryos, and pupae. Eight different transcripts are produced by the use of combinations of alternative promoters, exons, and splice acceptor sites; the various species are not all equally abundant. The 3'-most exon is unusual in that it is completely noncoding. These transcripts can potentially generate four protein isoforms that differ in their N-terminal 16 to 21 amino acids but are identical in the remainder of the protein, including the RNP consensus motif domain and the glycine-rich domain characteristic of the mammalian A1 protein. We suggest that these sequence differences could affect the affinities of the proteins for RNA or other protein components of heterogeneous nuclear RNP complexes, leading to differences in function. Images PMID:2104660

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

    PubMed

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

    2016-06-01

    Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism. PMID:27165283

  19. An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

    SciTech Connect

    Adamik, Barbara; Islam, Aminul; Rouhani, Farshid N.; Hawari, Feras I.; Zhang Jing; Levine, Stewart J.

    2008-07-04

    The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1{beta}-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1{beta}-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains.

  20. The host protein CLUH participates in the subnuclear transport of influenza virus ribonucleoprotein complexes.

    PubMed

    Ando, Tomomi; Yamayoshi, Seiya; Tomita, Yuriko; Watanabe, Shinji; Watanabe, Tokiko; Kawaoka, Yoshihiro

    2016-01-01

    The nucleus is highly compartmentalized yet dynamic. Subnuclear functions are regulated by controlling the subnuclear localization of the nuclear proteins. Influenza viral ribonucleoprotein (vRNP) is replicated in the nucleus and then exported to the cytoplasm. However, the precise subnuclear localization and transport of vRNPs remain unclear. Here, we show that CLUH, a host protein whose cellular function is not well established, plays a key role in the subnuclear transport of vRNP. Viral PB2 and M1 induced CLUH translocation to the nucleoplasm and SC35-positive speckles, respectively, even though CLUH is usually cytoplasmic. CLUH depletion inhibited the translocation of M1 to SC35-positive speckles, but did not interfere with PB2 localization to the nucleoplasm and disrupted the subnuclear transport of vRNP, abolishing vRNP nuclear export without affecting viral RNA or protein expression. Our findings suggest that CLUH plays a role in the subnuclear transport of progeny vRNP. PMID:27573102

  1. The algal metabolite yessotoxin affects heterogeneous nuclear ribonucleoproteins in HepG2 cells.

    PubMed

    Young, Clifford; Truman, Penelope; Boucher, Magalie; Keyzers, Robert A; Northcote, Peter; Jordan, T William

    2009-05-01

    The dinoflagellate metabolite yessotoxin (YTX) is produced by several species of algae and accumulates in marine food chains, leading to concerns about possible affects on aquaculture industries and human health. In mice used for toxicity testing, YTX is lethal by the intraperitoneal route, but is considerably less toxic when orally administered. The mode of action of YTX and its potential effect on humans is unclear and we therefore conducted the first proteomic analysis of the effects of this compound. We used 2-DE to examine protein changes in HepG2 cell cultures exposed to 1.4 microM YTX for 3, 12.5, 18 and 24 h. After selecting proteins that changed more than three-fold after YTX exposure, 55 spots were deemed significantly affected by the toxin (p<0.05). Major groups of affected proteins include members from the heterogeneous nuclear ribonucleoprotein (hnRNP), lamin, cathepsin and heat shock protein families that often are associated with apoptosis. We therefore confirmed apoptosis using Annexin-V-FLUOS staining of phosphatidylserine exposed at the surface of apoptotic cells. Ingenuity pathways analysis also indicated effects on pathways involved in protein processing, cell cycling and cell death. PMID:19343718

  2. Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP.

    PubMed

    Lemieux, Bruno; Laterreur, Nancy; Perederina, Anna; Noël, Jean-François; Dubois, Marie-Line; Krasilnikov, Andrey S; Wellinger, Raymund J

    2016-05-19

    Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs. PMID:27156450

  3. Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus

    NASA Astrophysics Data System (ADS)

    Zhou, Zhuo; Cao, Mengmeng; Guo, Yang; Zhao, Lili; Wang, Jingfeng; Jia, Xue; Li, Jianguo; Wang, Conghui; Gabriel, Gülsah; Xue, Qinghua; Yi, Yonghong; Cui, Sheng; Jin, Qi; Wang, Jianwei; Deng, Tao

    2014-02-01

    The ribonucleoprotein (RNP) of the influenza A virus is responsible for the transcription and replication of viral RNA in the nucleus. These processes require interplay between host factors and RNP components. Here, we report that the Fragile X mental retardation protein (FMRP) targets influenza virus RNA synthesis machinery and facilitates virus replication both in cell culture and in mice. We demonstrate that FMRP transiently associates with viral RNP and stimulates viral RNP assembly through RNA-mediated interaction with the nucleoprotein. Furthermore, the KH2 domain of FMRP mediates its association with the nucleoprotein. A point mutation (I304N) in the KH2 domain, identified from a Fragile X syndrome patient, disrupts the FMRP-nucleoprotein association and abolishes the ability of FMRP to participate in viral RNP assembly. We conclude that FMRP is a critical host factor used by influenza viruses to facilitate viral RNP assembly. Our observation reveals a mechanism of influenza virus RNA synthesis and provides insights into FMRP functions.

  4. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.

    PubMed

    Park, Ki-Eun; Park, Chi-Hun; Powell, Anne; Martin, Jessica; Donovan, David M; Telugu, Bhanu P

    2016-01-01

    The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals. PMID:27240344

  5. Large-scale immunopurification of ribonucleoprotein complexes from Drosophila nucleoplasmic extracts for tiling microarrays.

    PubMed

    Rio, Donald C

    2014-03-01

    It is of interest to be able to define sets of cellular RNAs associated with specific RNA-binding proteins. This "guilt by association" can lead to new insights into how RNA-binding proteins modulate posttranscriptional gene expression of specific target RNAs. To identify these RNAs, antibodies against RNA-binding proteins can be used to immunopurify endogenous RNA-protein complexes from cells, and then the associated RNAs can be characterized. The method described here was developed to identify binding regions on nuclear transcripts for Drosophila heterogeneous nuclear ribonucleoproteins (hnRNPs). An antibody is added to an RNP extract and incubated to allow antigen-antibody complexes to form. The antibody-antigen complexes are then retrieved by binding of the antibody constant region to staphylococcal Protein A immobilized on Sepharose beads. The bead-immobilized complexes are then washed and RNA is prepared. The RNA is used to generate random-primed cDNA, cRNA, and biotinlyated cDNA probes for use on Affymetrix whole-genome Drosophila tiling arrays. PMID:24591694

  6. A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

    PubMed

    Na, Hong; Laver, John D; Jeon, Jouhyun; Singh, Fateh; Ancevicius, Kristin; Fan, Yujie; Cao, Wen Xi; Nie, Kun; Yang, Zhenglin; Luo, Hua; Wang, Miranda; Rissland, Olivia; Westwood, J Timothy; Kim, Philip M; Smibert, Craig A; Lipshitz, Howard D; Sidhu, Sachdev S

    2016-04-01

    Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins. PMID:26847261

  7. Role and molecular mechanism of heterogeneous nuclear ribonucleoprotein K in tumor development and progression

    PubMed Central

    LU, JING; GAO, FENG-HOU

    2016-01-01

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the hnRNP family, which exists in the nucleus and the cytoplasm simultaneously. It is a multifunctional protein that can participate in a variety of regulatory progressions of gene expression and signal transduction, such as chromatin remodeling, transcription, RNA alternative splicing and translation. hnRNP K not only directly binds to the kinases, but also recruits the associated factors regarding transcription, splicing and translation to control gene expression, and therefore, it serves as a docking platform for integrating transduction pathways to nucleic acid-directed processes. Numerous studies also show that abnormal expression of hnRNP K is closely associated with the tumor formation. This protein is overexpressed in numerous types of cancer and its aberrant cytoplasmic localization is also associated with a worse prognosis for patients. These results consistently indicate that hnRNP K has a key role in cancer progression. To understand the hnRNP K pathophysiological process in tumor disease, the previous research results regarding the association between hnRNP K and tumors were reviewed. PMID:27284403

  8. 3' UTR length and messenger ribonucleoprotein composition determine endocleavage efficiencies at termination codons.

    PubMed

    Boehm, Volker; Haberman, Nejc; Ottens, Franziska; Ule, Jernej; Gehring, Niels H

    2014-10-23

    Nonsense-mediated mRNA decay (NMD) degrades different classes of mRNAs, including transcripts with premature termination codons (PTCs). The NMD factor SMG6 initiates degradation of substrate mRNAs by endonucleolytic cleavage. Here, we aim to delineate the cascade of NMD-activating events that culminate in endocleavage. We report that long 3' UTRs elicit SMG6-mediated endonucleolytic degradation. The presence of an exon-junction complex (EJC) within the 3' UTR strongly stimulates endocleavage in a distance-independent manner. The interaction of SMG6 with EJCs is not required for endocleavage. Whereas the core NMD component UPF2 supports endonucleolytic decay of long 3' UTR mRNAs, it is mostly dispensable during EJC-stimulated endocleavage. Using high-throughput sequencing, we map endocleavage positions of different PTC-containing reporter mRNAs and an endogenous NMD substrate to regions directly at and downstream of the termination codon. These results reveal how messenger ribonucleoprotein (mRNP) parameters differentially influence SMG6-executed endonucleolysis and uncover central characteristics of this phenomenon associated with translation termination. PMID:25310981

  9. LRPPRC and SLIRP interact in a ribonucleoprotein complex that regulates posttranscriptional gene expression in mitochondria.

    PubMed

    Sasarman, Florin; Brunel-Guitton, Catherine; Antonicka, Hana; Wai, Timothy; Shoubridge, Eric A

    2010-04-15

    Mutations in LRPPRC are responsible for the French Canadian variant of Leigh syndrome (LSFC), a neurodegenerative disorder caused by a tissue-specific deficiency in cytochrome c oxidase (COX). To investigate the pathogenic mechanism of disease, we studied LRPPRC function in LSFC and control fibroblasts. The level of mutated LRPPRC is reduced in LSFC cells, and this results in decreased steady-state levels of most mitochondrial mRNAs, but not rRNAs or tRNAs, a phenotype that can be reproduced by siRNA-mediated knockdown of LRPPRC in control cells. Processing of the primary transcripts appears normal. The resultant defect in mitochondrial protein synthesis in LSFC cells disproportionately affects the COX subunits, leading to an isolated COX assembly defect. Further knockdown of LRPPRC produces a generalized assembly defect in all oxidative phosphorylation complexes containing mtDNA-encoded subunits, due to a severe decrease in all mitochondrial mRNAs. LRPPRC exists in a high-molecular-weight complex, and it coimmunoprecipitates with SLIRP, a stem-loop RNA-binding protein. Although this interaction does not depend on mitochondrial mRNA, both proteins show reduced stability in its absence. These results implicate LRPPRC in posttranscriptional mitochondrial gene expression as part of a ribonucleoprotein complex that regulates the stability and handling of mature mRNAs. PMID:20200222

  10. Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos.

    PubMed

    Zhang, Ping; Wang, Ningling; Lin, Xianhua; Jin, Li; Xu, Hong; Li, Rong; Huang, Hefeng

    2016-02-26

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. PMID:26850853

  11. Detection of mRNA sequences in nuclear 30S ribonucleoprotein subcomplexes.

    PubMed Central

    Kinniburgh, A J; Martin, T E

    1976-01-01

    RNA from nuclear 30S ribonucleoprotein (RNP) complexes of mouse ascites cells has been shows to contain sequences homologous to poly(A) + mRNA by its ability to hybridize with complementary DNA prepared from poly(A) + mRNA template. Analysis of the hybridization kinetics of poly(A) + mRNA with its own complementary DNA revealed several abundancy classes. The total complexity of poly(A) + mRNA from ascites cells was estimated to be approximately 30,000 sequences of average molecular weight (6 X 10(5)). When the hybridization reaction of 30S RNP-RNA with mRNA-specific cDNA was compared to the homologous reaction the majority, and most probably all, of the poly(A) + mRNA sequences were found to be present in the RNA. The kinetics of hybridization suggest that 10-15% of the RNA in this RNP complex is homologous to poly(A) + mRNA. The 30S RNP subcomplexes therefore contain nuclear poly(A) + mRNA sequences as well as the bulk of heterogeneous RNA. PMID:1066686

  12. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

    PubMed

    Bai, Bing; Hales, Chadwick M; Chen, Ping-Chung; Gozal, Yair; Dammer, Eric B; Fritz, Jason J; Wang, Xusheng; Xia, Qiangwei; Duong, Duc M; Street, Craig; Cantero, Gloria; Cheng, Dongmei; Jones, Drew R; Wu, Zhiping; Li, Yuxin; Diner, Ian; Heilman, Craig J; Rees, Howard D; Wu, Hao; Lin, Li; Szulwach, Keith E; Gearing, Marla; Mufson, Elliott J; Bennett, David A; Montine, Thomas J; Seyfried, Nicholas T; Wingo, Thomas S; Sun, Yi E; Jin, Peng; Hanfelt, John; Willcock, Donna M; Levey, Allan; Lah, James J; Peng, Junmin

    2013-10-01

    Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis. PMID:24023061

  13. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins

    PubMed Central

    Park, Ki-Eun; Park, Chi-Hun; Powell, Anne; Martin, Jessica; Donovan, David M.; Telugu, Bhanu P.

    2016-01-01

    The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals. PMID:27240344

  14. Heterogeneous nuclear ribonucleoprotein K is overexpressed and associated with poor prognosis in gastric cancer.

    PubMed

    Yang, Ruirui; Zeng, Ying; Xu, Haifan; Chen, Zhuo; Xiang, Mengqin; Fu, Yun; Yin, Yufang; Zhong, Jing; Zeng, Min; Wang, Peihua; You, Qin; Zeng, Xi

    2016-08-01

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is one of the major pre-mRNA-binding proteins, that is involved in translational modifications. In our previous studies, we found that hnRNP K is associated with human gastric cancer. The protein levels of hnRNP K were detected in cell lines and tissue microarrays. The correlation between hnRNP K expression and patient survival rate was evaluated by Kaplan-Meier survival analysis. In addition, we also detected hnRNP K expression in preoperative and postoperative serum samples from patients with gastric cancer, and serum samples from healthy volunteers. We found that hnRNP K was overexpressed in the gastric cancer cell lines. The levels of hnRNP K were significantly elevated in the gastric cancer tissues compared with that noted in the tumor-adjacent gastric mucosal and normal gastric mucosal sampes, and hnRNP K expression was found to correlate with tumor stage and lymph node metastasis. However, the level of serum hnRNP K did not differ significantly between gastric cancer patients and healthy volunteers. We also found that patients whose tumors showed elevated expression of hnRNP K had poor survival. The present study suggests that hnRNP K is a promising tissue biomarker for diagnosing gastric cancer and is a prognostic indicator for patients with gastric cancer. PMID:27278897

  15. Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.

    PubMed

    Chen, Sean; Lee, Benjamin; Lee, Angus Yiu-Fai; Modzelewski, Andrew J; He, Lin

    2016-07-01

    The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals. PMID:27151215

  16. Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization

    PubMed Central

    Ariza, Antonio; Tanner, Sian J.; Walter, Cheryl T.; Dent, Kyle C.; Shepherd, Dale A.; Wu, Weining; Matthews, Susan V.; Hiscox, Julian A.; Green, Todd J.; Luo, Ming; Elliott, Richard M.; Fooks, Anthony R.; Ashcroft, Alison E.; Stonehouse, Nicola J.; Ranson, Neil A.; Barr, John N.; Edwards, Thomas A.

    2013-01-01

    All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses. PMID:23595147

  17. K-homology Nuclear Ribonucleoproteins Regulate Floral Organ Identity and Determinacy in Arabidopsis

    PubMed Central

    Rodríguez-Cazorla, Encarnación; Ripoll, Juan José; Andújar, Alfonso; Bailey, Lindsay J.; Martínez-Laborda, Antonio; Yanofsky, Martin F.; Vera, Antonio

    2015-01-01

    Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process. PMID:25658099

  18. Effects of stress and aging on ribonucleoprotein assembly and function in the germ line

    PubMed Central

    Schisa, Jennifer A.

    2016-01-01

    In a variety of cell types, ribonucleoprotein (RNP) complexes play critical roles in regulating RNA metabolism. The germ line contains RNPs found also in somatic cells, such as processing (P) bodies and stress granules, as well as several RNPs unique to the germ line, including germ granules, nuage, Balbiani bodies, P granules, U bodies, and sponge bodies. Recent advances have identified a conserved response of germ line RNPs to environmental stresses such as nutritional stress and heat shock. The RNPs increase significantly in size based on cytology; their morphology and subcellular localization changes, and their composition changes. These dynamic changes are reversible when stresses diminish, and similar changes occur in response to aging or extended meiotic arrest prior to fertilization of oocytes. Intriguing correlations exist between the dynamics of the RNPs and the microtubule cytoskeleton and its motor proteins, suggesting a possible mechanism for the assembly and dissociation of the large RNP granules. Similarly, coordinated changes of the nuclear membrane and endoplasmic reticulum may also help unravel the regulatory mechanisms of RNP dynamics. Based on their composition, the RNPs are thought to regulate mRNA decay and/or translation, and initial support for some of these roles is now at hand. Ultimately, the question of why RNP remodeling occurs to such a large extent during a variety of stresses and aging remains to be fully answered, but a current attractive hypothesis is that the plasticity promotes the maintenance of oocyte quality. PMID:24523207

  19. Largest-ever Ozone Hole over Antarctica

    NASA Technical Reports Server (NTRS)

    2002-01-01

    A NASA instrument has detected an Antarctic ozone 'hole' (what scientists call an 'ozone depletion area') that is three times larger than the entire land mass of the United States-the largest such area ever observed. The 'hole' expanded to a record size of approximately 11 million square miles (28.3 million square kilometers) on Sept. 3, 2000. The previous record was approximately 10.5 million square miles (27.2 million square km) on Sept. 19, 1998. The ozone hole's size currently has stabilized, but the low levels in its interior continue to fall. The lowest readings in the ozone hole are typically observed in late September or early October each year. 'These observations reinforce concerns about the frailty of Earth's ozone layer. Although production of ozone-destroying gases has been curtailed under international agreements, concentrations of the gases in the stratosphere are only now reaching their peak. Due to their long persistence in the atmosphere, it will be many decades before the ozone hole is no longer an annual occurrence,' said Dr. Michael J. Kurylo, manager of the Upper Atmosphere Research Program, NASA Headquarters, Washington, DC. Ozone molecules, made up of three atoms of oxygen, comprise a thin layer of the atmosphere that absorbs harmful ultraviolet radiation from the Sun. Most atmospheric ozone is found between approximately six miles (9.5 km) and 18 miles (29 km) above the Earth's surface. Scientists continuing to investigate this enormous hole are somewhat surprised by its size. The reasons behind the dimensions involve both early-spring conditions, and an extremely intense Antarctic vortex. The Antarctic vortex is an upper-altitude stratospheric air current that sweeps around the Antarctic continent, confining the Antarctic ozone hole. 'Variations in the size of the ozone hole and of ozone depletion accompanying it from one year to the next are not unexpected,' said Dr. Jack Kaye, Office of Earth Sciences Research Director, NASA Headquarters

  20. Spitzer/IRAC Photometry Of The Four Largest Uranian Satellites

    NASA Astrophysics Data System (ADS)

    Cartwright, Richard; Emery, J.; Rivkin, A.; Trilling, D.

    2012-10-01

    The surfaces of the four largest Uranian satellites are dominated by water ice and a spectrally neutral constituent that is likely carbonaceous in composition. CO2 ice has been detected on Ariel, Umbriel, and Titania, with no detection on the furthest regular Uranian satellite, Oberon (Grundy et al., 2003, 2006). Whether CO2 ice is primordial or is actively produced in the Uranian system is unclear; however, it seems unlikely that primordial CO2 ice would remain exposed on an icy satellite surface over the age of the Solar System. One possible mechanism for producing CO2 ice is bombardment of water ice and carbonaceous material by charged particles caught in Uranus’ magnetic field. Unlike the other large Uranian satellites, Oberon spends part of its orbit outside the confines of Uranus’ magnetic field, which might help explain why CO2 ice has yet to be detected on Oberon. We are using photometric data gathered by the Infrared Array Camera (IRAC), onboard the Spitzer Space Telescope (SST), in order to search for the signature of CO2 ice on Oberon, and confirm its presence on Ariel, Umbriel, and Titania at longer wavelengths than previous studies. IRAC collects data in four different channels, which are centered roughly at 3.6, 4.5, 5.8, and 8.0 µm. Additionally, we are gathering spectroscopic data using SpeX on IRTF, at similar longitudes to the IRAC observations, in order to characterize the distribution of CO2 ice on these icy satellites over a wide range of near-infrared wavelengths. Our preliminary photometry results for Oberon indicate that there is a steep reduction in reflected solar flux from channel 1 to channel 2, suggesting that surface materials are absorbing photons at wavelengths within the bandpass of channel 2. We will present the results of our photometric analysis of the four largest Uranian moons.

  1. First Light for World's Largest 'Thermometer Camera'

    NASA Astrophysics Data System (ADS)

    2007-08-01

    LABOCA in Service at APEX The world's largest bolometer camera for submillimetre astronomy is now in service at the 12-m APEX telescope, located on the 5100m high Chajnantor plateau in the Chilean Andes. LABOCA was specifically designed for the study of extremely cold astronomical objects and, with its large field of view and very high sensitivity, will open new vistas in our knowledge of how stars form and how the first galaxies emerged from the Big Bang. ESO PR Photo 35a/07 ESO PR Photo 35a/07 LABOCA on APEX "A large fraction of all the gas in the Universe has extremely cold temperatures of around minus 250 degrees Celsius, a mere 20 degrees above absolute zero," says Karl Menten, director at the Max Planck Institute for Radioastronomy (MPIfR) in Bonn, Germany, that built LABOCA. "Studying these cold clouds requires looking at the light they radiate in the submillimetre range, with very sophisticated detectors." Astronomers use bolometers for this task, which are, in essence, thermometers. They detect incoming radiation by registering the resulting rise in temperature. More specifically, a bolometer detector consists of an extremely thin foil that absorbs the incoming light. Any change of the radiation's intensity results in a slight change in temperature of the foil, which can then be registered by sensitive electronic thermometers. To be able to measure such minute temperature fluctuations requires the bolometers to be cooled down to less than 0.3 degrees above absolute zero, that is below minus 272.85 degrees Celsius. "Cooling to such low temperatures requires using liquid helium, which is no simple feat for an observatory located at 5100m altitude," says Carlos De Breuck, the APEX instrument scientist at ESO. Nor is it simple to measure the weak temperature radiation of astronomical objects. Millimetre and submillimetre radiation opens a window into the enigmatic cold Universe, but the signals from space are heavily absorbed by water vapour in the Earth

  2. Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes under Hypoxia Stress.

    PubMed

    Lokdarshi, Ansul; Conner, W Craig; McClintock, Carlee; Li, Tian; Roberts, Daniel M

    2016-02-01

    During waterlogging and the associated oxygen deprivation stress, plants respond by the induction of adaptive programs, including the redirected expression of gene networks toward the synthesis of core hypoxia-response proteins. Among these core response proteins in Arabidopsis (Arabidopsis thaliana) is the calcium sensor CML38, a protein related to regulator of gene silencing calmodulin-like proteins (rgsCaMs). CML38 transcripts are up-regulated more than 300-fold in roots within 6 h of hypoxia treatment. Transfer DNA insertional mutants of CML38 show an enhanced sensitivity to hypoxia stress, with lowered survival and more severe inhibition of root and shoot growth. By using yellow fluorescent protein (YFP) translational fusions, CML38 protein was found to be localized to cytosolic granule structures similar in morphology to hypoxia-induced stress granules. Immunoprecipitation of CML38 from the roots of hypoxia-challenged transgenic plants harboring CML38pro::CML38:YFP followed by liquid chromatography-tandem mass spectrometry analysis revealed the presence of protein targets associated with messenger RNA ribonucleoprotein (mRNP) complexes including stress granules, which are known to accumulate as messenger RNA storage and triage centers during hypoxia. This finding is further supported by the colocalization of CML38 with the mRNP stress granule marker RNA Binding Protein 47 (RBP47) upon cotransfection of Nicotiana benthamiana leaves. Ruthenium Red treatment results in the loss of CML38 signal in cytosolic granules, suggesting that calcium is necessary for stress granule association. These results confirm that CML38 is a core hypoxia response calcium sensor protein and suggest that it serves as a potential calcium signaling target within stress granules and other mRNPs that accumulate during flooding stress responses. PMID:26634999

  3. Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules.

    PubMed

    Shively, Christian A; Kweon, Hye Kyong; Norman, Kaitlyn L; Mellacheruvu, Dattatreya; Xu, Tao; Sheidy, Daniel T; Dobry, Craig J; Sabath, Ivan; Cosky, Eric E P; Tran, Elizabeth J; Nesvizhskii, Alexey; Andrews, Philip C; Kumar, Anuj

    2015-10-01

    Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs. PMID:26447709

  4. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.

    PubMed

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R; Subramaniam, Meena; Gate, Rachel E; Haliburton, Genevieve E; Ye, Chun J; Bluestone, Jeffrey A; Doudna, Jennifer A; Marson, Alexander

    2015-08-18

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells. PMID:26216948

  5. Ribonomic analysis of human DZIP1 reveals its involvement in ribonucleoprotein complexes and stress granules

    PubMed Central

    2014-01-01

    Background DZIP1 (DAZ-interacting protein 1) has been described as a component of the Hh signaling pathway with a putative regulatory role in ciliogenesis. DZIP1 interacts with DAZ RNA binding proteins in embryonic stem cells and human germ cells suggesting a role in mRNA regulation. Results We investigated DZIP1 function in HeLa cells and its involvement in ribonucleoprotein complexes. DZIP1 was predominantly located in granules in the cytoplasm. Under oxidative stress conditions, DZIP1 re-localized to stress granules. DZIP appears to be important for the formation of stress granules during the stress response. We used immunoprecipitation assays with antibodies against DZIP1 and microarray hybridization to identify mRNAs associated with DZIP1. The genetic networks formed by the DZIP1-associated mRNAs were involved in cell cycle and gene expression regulation. DZIP1 is involved in the Hedgehog signaling pathway. We used cyclopamine, a specific inhibitor of this pathway, to analyze the expression of DZIP1 and its associated mRNAs. The abundance of DZIP1-associated mRNAs increased with treatment; however, the silencing or overexpression of DZIP1 in HeLa cells had no effect on the accumulation of the associated mRNAs. Polysomal profile analysis by sucrose gradient centrifugation demonstrated the presence of DZIP1 in the polysomal fraction. Conclusions Our results suggest that DZIP1 is part of an RNP complex that occupies various subcellular locations. The diversity of the mRNAs associated with DZIP1 suggests that this protein is a component of different RNPs associated with translating polysomes and with RNA granules. PMID:24993635

  6. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    PubMed Central

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; Marson, Alexander

    2015-01-01

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells. PMID:26216948

  7. Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex.

    PubMed

    Chu, E; Copur, S M; Ju, J; Chen, T M; Khleif, S; Voeller, D M; Mizunuma, N; Patel, M; Maley, G F; Maley, F; Allegra, C J

    1999-02-01

    A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression. PMID:9891091

  8. Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

    PubMed Central

    Chu, Edward; Copur, Sitki M.; Ju, Jingfang; Chen, Tian-men; Khleif, Samir; Voeller, Donna M.; Mizunuma, Nobuyuki; Patel, Mahendra; Maley, Gladys F.; Maley, Frank; Allegra, Carmen J.

    1999-01-01

    A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression. PMID:9891091

  9. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    DOE PAGESBeta

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; et al

    2015-07-27

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9more » RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

  10. Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules

    PubMed Central

    Mellacheruvu, Dattatreya; Xu, Tao; Sheidy, Daniel T.; Dobry, Craig J.; Sabath, Ivan; Cosky, Eric E. P.; Tran, Elizabeth J.; Nesvizhskii, Alexey; Andrews, Philip C.; Kumar, Anuj

    2015-01-01

    Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs. PMID:26447709

  11. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    SciTech Connect

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; Marson, Alexander

    2015-07-27

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

  12. Differential requirements for H/ACA ribonucleoprotein components in cell proliferation and response to DNA damage.

    PubMed

    Lin, Ping; Mobasher, Maral E; Hakakian, Yasaman; Kakarla, Veena; Naseem, Anum F; Ziai, Heliya; Alawi, Faizan

    2015-12-01

    H/ACA ribonucleoproteins (RNPs) are comprised of four conserved proteins, dyskerin, NHP2, NOP10, and GAR1, and a function-specifying, noncoding H/ACA RNA. H/ACA RNPs contribute to telomerase assembly and stabilization, and posttranscriptional processing of nascent ribosomal RNA and spliceosomal RNA. However, very little is known about the coordinated action of the four proteins in other biologic processes. As described herein, we observed a differential requirement for the proteins in cell proliferation and identified a possible reliance for these factors in regulation of specific DNA damage biomarkers. In particular, GAR1 expression was upregulated following exposure to all forms of genotoxic stress tested. In contrast, levels of the other proteins were either reduced or unaffected. Only GAR1 showed an altered subcellular localization with a shift from the nucleolus to the nucleoplasm after ultraviolet-C irradiation and doxorubicin treatments. Transient siRNA-mediated depletion of GAR1 and dyskerin arrested cell proliferation, whereas loss of either NHP2 or NOP10 had no effect. Finally, loss of dyskerin, GAR1, NHP2, and NOP10, respectively, limited the accumulation of DNA damage biomarkers. However, the individual responses were dependent upon the specific type of damage incurred. In general, loss of GAR1 had the most suppressive effect on the biomarkers tested. Since the specific responses to genotoxic stress, the contribution of each protein to cell proliferation, and the activation of DNA damage biomarkers were not equivalent, this suggests the possibility that at least some of the proteins, most notably GAR1, may potentially function independently of their respective roles within H/ACA RNP complexes. PMID:26265134

  13. Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes under Hypoxia Stress1[OPEN

    PubMed Central

    McClintock, Carlee; Li, Tian

    2016-01-01

    During waterlogging and the associated oxygen deprivation stress, plants respond by the induction of adaptive programs, including the redirected expression of gene networks toward the synthesis of core hypoxia-response proteins. Among these core response proteins in Arabidopsis (Arabidopsis thaliana) is the calcium sensor CML38, a protein related to regulator of gene silencing calmodulin-like proteins (rgsCaMs). CML38 transcripts are up-regulated more than 300-fold in roots within 6 h of hypoxia treatment. Transfer DNA insertional mutants of CML38 show an enhanced sensitivity to hypoxia stress, with lowered survival and more severe inhibition of root and shoot growth. By using yellow fluorescent protein (YFP) translational fusions, CML38 protein was found to be localized to cytosolic granule structures similar in morphology to hypoxia-induced stress granules. Immunoprecipitation of CML38 from the roots of hypoxia-challenged transgenic plants harboring CML38pro::CML38:YFP followed by liquid chromatography-tandem mass spectrometry analysis revealed the presence of protein targets associated with messenger RNA ribonucleoprotein (mRNP) complexes including stress granules, which are known to accumulate as messenger RNA storage and triage centers during hypoxia. This finding is further supported by the colocalization of CML38 with the mRNP stress granule marker RNA Binding Protein 47 (RBP47) upon cotransfection of Nicotiana benthamiana leaves. Ruthenium Red treatment results in the loss of CML38 signal in cytosolic granules, suggesting that calcium is necessary for stress granule association. These results confirm that CML38 is a core hypoxia response calcium sensor protein and suggest that it serves as a potential calcium signaling target within stress granules and other mRNPs that accumulate during flooding stress responses. PMID:26634999

  14. Protein and gene expression characteristics of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma

    PubMed Central

    Sun, Yu-Lin; Liu, Fei; Liu, Fang; Zhao, Xiao-Hang

    2016-01-01

    AIM To investigate the expression characteristics of heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) mRNA and protein in cell lines and tissues of esophageal squamous cell carcinoma (ESCC). METHODS Western blotting was used to assess the expression of HNRNPH1 protein in seven ESCC cell lines and 30 paired fresh tissue specimens. The subcellular localization of HNRNPH1 was determined by immunofluorescence in ESCC cells. The RNA sequencing data from 87 patients with ESCC were obtained from the cancer genome atlas (TCGA), and the expression and clinical characteristics analysis of different transcript variants of HNRNPH1 were evaluated in this dataset. In addition, immunohistochemistry was carried out to detect the expression of HNRNPH1 protein in 125 patients. RESULTS The expression of HNRNPH1 protein varied across different ESCC cell lines. It was exclusively restricted to the nucleus of the ESCC cells. There are two transcript variants of the HNRNPH1 gene. Variant 1 was constitutively expressed, and its expression did not change during tumorigenesis. In contrast, levels of variant 2 were low in non-tumorous tissues and were dramatically increased in ESCC (P = 0.0026). The high levels of variant 2 were associated with poorer differentiated tumors (P = 0.0287). Furthermore, in paired fresh tissue specimens, HNRNPH1 protein was overexpressed in 73.3% (22/30) of neoplastic tissues. HNRNPH1 was significantly upregulated in ESCC, with strong staining in 43.2% (54/125) of tumor tissues and 22.4% (28/125) of matched non-cancerous tissues (P = 0.0005). Positive HNRNPH1 expression was significantly associated with poor tumor differentiation degree (P = 0.0337). CONCLUSION The different alternative transcript variants of HNRNPH1 exhibited different expression changes during tumorigenesis. Its mRNA and protein were overexpressed in ESCC and associated with poorer differentiation of tumor cells. These findings highlight the potential of HNRNPH1 in the therapy and diagnosis

  15. Plans for building the largest thin solenoid ever

    SciTech Connect

    Heim, J.; Bowers, J.; Deis, G.

    1993-09-08

    The superconducting solenoid magnet for the GEM detector poses unusual fabrication and handling challenges because of its extraordinary size. It will be more than 30% larger in diameter than the largest existing particle detector coils. Each of the two coil elements that compose the air-core solenoid, will be about 19 meters in diameter and 15 meters long. Major components weighing as much as 1500 Mg must be transported and manipulated at the Interaction Region 5 (IR5) fabrication site of the SSC Laboratory as the magnets are fabricated. Because of their large size, the magnets will be fabricated, assembled and tested at special purpose facilities at the IR5 site. The site-use plan must accommodate the fabrication of other detector components and the assembly of large flux shaping iron structures in a timely manner to allow subsequent testing and detector assembly. Each cold mass will be composed of twelve 45-Mg coil windings that are joined prior to assembly into the 19-m diam annular cryostat.

  16. Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex

    PubMed Central

    Kiss, Gabriella; Holl, Jens M.; Williams, Grant M.; Alonas, Eric; Vanover, Daryll; Lifland, Aaron W.; Gudheti, Manasa; Guerrero-Ferreira, Ricardo C.; Nair, Vinod; Yi, Hong; Graham, Barney S.; Santangelo, Philip J.

    2014-01-01

    ABSTRACT Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. IMPORTANCE hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable

  17. Spinal muscular atrophy and a model for survival of motor neuron protein function in axonal ribonucleoprotein complexes.

    PubMed

    Rossoll, Wilfried; Bassell, Gary J

    2009-01-01

    Spinal muscular atrophy (SMA) is a neurodegenerative disease that results from loss of function of the SMN1 gene, encoding the ubiquitously expressed survival of motor neuron (SMN) protein, a protein best known for its housekeeping role in the SMN-Gemin multiprotein complex involved in spliceosomal small nuclear ribonucleoprotein (snRNP) assembly. However, numerous studies reveal that SMN has many interaction partners, including mRNA binding proteins and actin regulators, suggesting its diverse role as a molecular chaperone involved in mRNA metabolism. This review focuses on studies suggesting an important role of SMN in regulating the assembly, localization, or stability of axonal messenger ribonucleoprotein (mRNP) complexes. Various animal models for SMA are discussed, and phenotypes described that indicate a predominant function for SMN in neuronal development and synapse formation. These models have begun to be used to test different therapeutic strategies that have the potential to restore SMN function. Further work to elucidate SMN mechanisms within motor neurons and other cell types involved in neuromuscular circuitry hold promise for the potential treatment of SMA. PMID:19343312

  18. Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a novel oncotarget

    PubMed Central

    Qiu, Ya-Qi; Yang, Cheng-Wei; Lee, Yue-Zhi; Yang, Ruey-Bing; Lee, Chih-Hao; Hsu, Hsing-Yu; Chang, Chien-Chung; Lee, Shiow-Ju

    2015-01-01

    Tylophorine compounds have been the focus of drug development for decades. Tylophorine derivatives exhibit anti-cancer activities but their cellular targets remain unknown. We used a biotinylated tylophorine derivative to probe for the interacting cellular target(s) of tylophorine. Tylophorine directly binds to caprin-1 and consequently enhances the recruitment of G3BP1, c-Myc mRNA, and cyclin D2 mRNA to form a ribonucleoprotein complex. Subsequently, this tylophorine targeted ribonucleoprotein complex is sequestered to the polysomal fractions and the protein expressions of the associated mRNA-transcripts are repressed. Caprin-1 depleted carcinoma cells become more resistant to tylophorine, associated with decreased formation of the ribonucleoprotein complex targeted by tylophorine. Consequently, tylophorine downregulates c-Myc and cyclins D1/D2, causing hypophosphorylation of Rb and suppression of both processing-body formation and the Warburg effect. Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds. Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b. Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex. PMID:25669982

  19. Detail of the largest generator, an Enterprise 5000, also known ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Detail of the largest generator, an Enterprise 5000, also known as "Big Bertha", looking southeast - March Air Force Base, Strategic Air Command, Utility Building, 5220 Riverside Drive, Moreno Valley, Riverside County, CA

  20. Approximating the largest eigenvalue of network adjacency matrices

    NASA Astrophysics Data System (ADS)

    Restrepo, Juan G.; Ott, Edward; Hunt, Brian R.

    2007-11-01

    The largest eigenvalue of the adjacency matrix of a network plays an important role in several network processes (e.g., synchronization of oscillators, percolation on directed networks, and linear stability of equilibria of network coupled systems). In this paper we develop approximations to the largest eigenvalue of adjacency matrices and discuss the relationships between these approximations. Numerical experiments on simulated networks are used to test our results.

  1. Comparative Study of Two Box H/ACA Ribonucleoprotein Pseudouridine-Synthases: Relation between Conformational Dynamics of the Guide RNA, Enzyme Assembly and Activity

    PubMed Central

    Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10–L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21. PMID:23922977

  2. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.

    PubMed

    Paix, Alexandre; Folkmann, Andrew; Rasoloson, Dominique; Seydoux, Geraldine

    2015-09-01

    Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins. PMID:26187122

  3. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes

    PubMed Central

    Paix, Alexandre; Folkmann, Andrew; Rasoloson, Dominique; Seydoux, Geraldine

    2015-01-01

    Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro–assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins. PMID:26187122

  4. Time scale of the largest imaginable magnetic storm

    NASA Astrophysics Data System (ADS)

    Vasyliūnas, V. M.

    2013-01-01

    The depression of the horizontal magnetic field at Earth's equator for the largest imaginable magnetic storm has been estimated (Vasyliūnas, 2011a) as -Dst ~ 2500 nT, from the assumption that the total pressure in the magnetosphere (plasma plus magnetic field perturbation) is limited, in order of magnitude, by the minimum pressure of Earth's dipole field at the location of each flux tube. The obvious related question is how long it would take the solar wind to supply the energy content of this largest storm. The maximum rate of energy input from the solar wind to the magnetosphere can be evaluated on the basis either of magnetotail stress balance or of polar cap potential saturation, giving an estimate of the time required to build up the largest storm, which (for solar-wind and magnetospheric parameter values typical of observed superstorms) is roughly between ~2 and ~6 h.

  5. Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor.

    PubMed Central

    Ohlsson, R I; van Eekelen, C; Philipson, L

    1982-01-01

    Heterogeneous nuclear protein complexes (hnRNP) containing the precursor RNA from the adenovirus early region 2 were analysed to determine the specificity of protein-RNA interaction. RNA precursor sequences were present in isolated hnRNP complexes and endogenous 30S particles. At least 20-40 bases long fragments were protected when RNase A was used to remove unprotected RNA sequences in hnRNA complexes. Similarly around 40 bases of RNA were protected in 30S particles. These sequences represent discrete regions of the adenovirus genome. Especially sequences complementary to the EcoRI-F fragment encoding the first leader and the major intron for the DNA binding protein (DBP) RNA precursor, were analysed in detail. Tentatively, sequences resistant to RNase A were located in the middle of the intron and at the splice-donor junction of the first leader of the DBP precursor RNA. The same sequences were identified irrespective whether hnRNP complexes or 30S particles were used suggesting that 30S particles originate from hnRNP complexes. A 38.000 dalton protein appears to be in direct contact with RNA sequences complementary to the EcoRI-F fragment. Images PMID:6285286

  6. Building Earth's Largest Library: Driving into the Future.

    ERIC Educational Resources Information Center

    Coffman, Steve

    1999-01-01

    Examines the Amazon.com online bookstore as a blueprint for designing the world's largest library. Topics include selection; accessibility and convenience; quality of Web sites and search tools; personalized service; library collection development, including interlibrary loan; library catalogs and catalog records; a circulation system; costs;…

  7. Cassini sheds light on Titan's second largest lake, Ligeia Mare

    NASA Astrophysics Data System (ADS)

    Wendel, JoAnna

    2014-04-01

    Saturn's largest moon, Titan, is known for its dense, planet-like atmosphere and large lakes most likely made of methane and ethane. It has been suggested that Titan's atmosphere and surface are a model of early Earth. Since the early 2000s, NASA's Cassini space probe has been unlocking secrets of the distant moon.

  8. Afterglows from the largest explosions in the universe

    PubMed Central

    Hartmann, Dieter H.

    1999-01-01

    The distinction of “largest explosions in the universe” has been bestowed on cosmic gamma-ray bursts. Their afterglows are brighter than supernovae and therefore are called hypernovae. Photometry and spectroscopy of these afterglows have provided major breakthroughs in our understanding of this mysterious phenomenon. PMID:10220364

  9. The Power of Nature. World's Largest Math Event 5.

    ERIC Educational Resources Information Center

    National Council of Teachers of Mathematics, Inc., Reston, VA.

    The theme of the fifth annual World's Largest Math Event (WLME 5) is "The Power of Nature." This theme encourages students to explore natural forces that affect humankind, including phenomena such as hurricanes, earthquakes, and snowstorms, and the mathematics that underlies their study. The 15 activities for WLMES have been grouped into five…

  10. The Single Largest Education Donor Comes with Controversy

    ERIC Educational Resources Information Center

    Finkel, Ed

    2012-01-01

    For the last decade, in districts big and small, the Bill & Melinda Gates Foundation has emerged as the largest private funder of educational efforts. This began with an initiative around small schools in the early to mid-2000s, mostly abandoned now, and has gained traction in the past few years in areas such as teacher evaluation, the Common Core…

  11. The Roper Center: The World's Largest Archive of Survey Data.

    ERIC Educational Resources Information Center

    Barry, John M.

    1988-01-01

    Describes the holdings and services provided by the Roper Center, the largest archive of survey data in the world. Explains the structure of and topics included in the online database, and discusses fees, membership opportunities, and future plans for the center. A sidebar by Burns W. Roper is included. (CLB)

  12. Animals as Our Companions: World's Largest Math Event 6.

    ERIC Educational Resources Information Center

    National Council of Teachers of Mathematics, Inc., Reston, VA.

    The World's Largest Math Event 6 (WLME 6) is the National Council of Teachers of Mathematics' (NCTM) celebration of mathematics and mathematics education and is the highlight of Mathematics Education Month. This year's event, slated for April 28, 2000, features the theme "Animals as Our Companions". This theme encourages students to explore the…

  13. Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors.

    PubMed

    Vale-Costa, Sílvia; Alenquer, Marta; Sousa, Ana Laura; Kellen, Bárbara; Ramalho, José; Tranfield, Erin M; Amorim, Maria João

    2016-04-15

    Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle. PMID:26940915

  14. Bayesian Predictive Distribution for the Magnitude of the Largest Aftershock

    NASA Astrophysics Data System (ADS)

    Shcherbakov, R.

    2014-12-01

    Aftershock sequences, which follow large earthquakes, last hundreds of days and are characterized by well defined frequency-magnitude and spatio-temporal distributions. The largest aftershocks in a sequence constitute significant hazard and can inflict additional damage to infrastructure. Therefore, the estimation of the magnitude of possible largest aftershocks in a sequence is of high importance. In this work, we propose a statistical model based on Bayesian analysis and extreme value statistics to describe the distribution of magnitudes of the largest aftershocks in a sequence. We derive an analytical expression for a Bayesian predictive distribution function for the magnitude of the largest expected aftershock and compute the corresponding confidence intervals. We assume that the occurrence of aftershocks can be modeled, to a good approximation, by a non-homogeneous Poisson process with a temporal event rate given by the modified Omori law. We also assume that the frequency-magnitude statistics of aftershocks can be approximated by Gutenberg-Richter scaling. We apply our analysis to 19 prominent aftershock sequences, which occurred in the last 30 years, in order to compute the Bayesian predictive distributions and the corresponding confidence intervals. In the analysis, we use the information of the early aftershocks in the sequences (in the first 1, 10, and 30 days after the main shock) to estimate retrospectively the confidence intervals for the magnitude of the expected largest aftershocks. We demonstrate by analysing 19 past sequences that in many cases we are able to constrain the magnitudes of the largest aftershocks. For example, this includes the analysis of the Darfield (Christchurch) aftershock sequence. The proposed analysis can be used for the earthquake hazard assessment and forecasting associated with the occurrence of large aftershocks. The improvement in instrumental data associated with early aftershocks can greatly enhance the analysis and

  15. Largest Lyapunov exponents for lattices of interacting classical spins.

    PubMed

    de Wijn, A S; Hess, B; Fine, B V

    2012-07-20

    We investigate how generic the onset of chaos in interacting many-body classical systems is in the context of lattices of classical spins with nearest-neighbor anisotropic couplings. Seven large lattices in different spatial dimensions were considered. For each lattice, more than 2000 largest Lyapunov exponents for randomly sampled Hamiltonians were numerically computed. Our results strongly suggest the absence of integrable nearest-neighbor Hamiltonians for the infinite lattices except for the trivial Ising case. In the vicinity of the Ising case, the largest Lyapunov exponents exhibit a power-law growth, while further away they become rather weakly sensitive to the Hamiltonian anisotropy. We also provide an analytical derivation of these results. PMID:22861854

  16. World's largest TLP moves onto deepwater Norwegian location

    SciTech Connect

    Vielvoye, R.

    1992-05-04

    This paper reports that the world's largest and most sophisticated tension leg platform (TLP) was floated out to Snorre oil field in the Norwegian North Sea last month. The 78,000 ton unit built by Norwegian independent, Sega Petroleum AS, Oslo, was installed in the southern part of block 34/7 and should produce first oil in August, about a month ahead of schedule.

  17. Blueprint of the world`s largest uranium markets

    SciTech Connect

    Bizal, M.R.

    1996-06-01

    This article is a review of two recently released reports: (1) the EURATOM Supply Agency Annual Report for 1995, and (2) the US Energy Information Administration Uranium Industry Annual for 1995. These reports provide myraid information on uranium production, deliveries, contracts, and prices, as well as enrichment market activity during 1995 in the world`s largest nuclear fuel markets: the European Union and the United States.

  18. Rare gas record in the largest Apollo 15 rock.

    NASA Technical Reports Server (NTRS)

    Marti, K.; Lightner, B. D.

    1972-01-01

    The results obtained from mass-spectrometry analyses of the noble gases He, Ne, Ar, Kr, and Xe in a 182-mg chip of the largest Apollo 15 rock 15555 are presented. The spallation krypton data indicate a well-shielded location through most of the time during which the rock was exposed to cosmic rays. Gas retention ages are estimated. No evidence for the presence of products from plutonium-244 or iodine-129 was found.

  19. Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.

    PubMed

    Cassar, Paul A; Carpenedo, Richard L; Samavarchi-Tehrani, Payman; Olsen, Jonathan B; Park, Chang Jun; Chang, Wing Y; Chen, Zhaoyi; Choey, Chandarong; Delaney, Sean; Guo, Huishan; Guo, Hongbo; Tanner, R Matthew; Perkins, Theodore J; Tenenbaum, Scott A; Emili, Andrew; Wrana, Jeffrey L; Gibbings, Derrick; Stanford, William L

    2015-10-01

    In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN. PMID:26265008

  20. Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells

    PubMed Central

    Gaudreau, Marie-Claude; Grapton, Damien; Helness, Anne; Vadnais, Charles; Fraszczak, Jennifer; Shooshtarizadeh, Peiman; Wilhelm, Brian; Robert, François; Heyd, Florian; Möröy, Tarik

    2016-01-01

    The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways. PMID:27271479

  1. The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs.

    PubMed Central

    Takagaki, Y; MacDonald, C C; Shenk, T; Manley, J L

    1992-01-01

    Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. We have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-crosslinked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. We have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long alpha-helix stabilized by salt bridges is predicted. An approximately 270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (approximately 20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs. Images PMID:1741396

  2. Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells.

    PubMed

    Gaudreau, Marie-Claude; Grapton, Damien; Helness, Anne; Vadnais, Charles; Fraszczak, Jennifer; Shooshtarizadeh, Peiman; Wilhelm, Brian; Robert, François; Heyd, Florian; Möröy, Tarik

    2016-01-01

    The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin(-)c-Kit(+) fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways. PMID:27271479

  3. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    SciTech Connect

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo . E-mail: sueokae@post.saga-med.ac.jp

    2005-08-05

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

  4. Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L.

    PubMed

    Lindsey, J William; deGannes, Samantha L; Pate, Kimberly A; Zhao, Xiurong

    2016-01-01

    Epstein-Barr virus (EBV) is associated with multiple sclerosis (MS), and antibodies to the EBV nuclear antigen-1 (EBNA-1) are consistently increased in MS patients. The hypothesis of this study is that anti-EBNA-1 antibodies cross-react with a self antigen in MS patients. We affinity purified anti-EBNA-1 antibodies from human plasma, used the anti-EBNA-1 to immunoprecipitate antigens from human brain, and identified bound antigens with mass spectrometry. Anti-EBNA-1 consistently bound heterogeneous nuclear ribonucleoprotein L (HNRNPL). We expressed both the long and short isoforms of this protein, and verified with Western blots and ELISA that the long isoform cross-reacts with EBNA-1. Immunohistochemistry demonstrated that anti-EBNA-1 bound to an antigen in the nucleus of cultured rat central nervous system cells. ELISA demonstrated the presence of antibodies to HNRNPL in the plasma of both healthy controls and MS patients, but anti-HNRNPL was not increased in MS patients. We conclude that HNRNPL is an autoantigen which cross-reacts with EBNA-1. The relevance of this autoantigen to MS and other autoimmune diseases remains to be investigated. PMID:26637929

  5. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

    PubMed Central

    Kim, Sojung; Kim, Daesik; Cho, Seung Woo; Kim, Jungeun; Kim, Jin-Soo

    2014-01-01

    RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection. PMID:24696461

  6. RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

    PubMed Central

    2014-01-01

    Background Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. Results We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. Conclusions This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism. PMID:24393626

  7. U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7.

    PubMed

    Savarese, Emina; Chae, Ohk-wha; Trowitzsch, Simon; Weber, Gert; Kastner, Berthold; Akira, Shizuo; Wagner, Hermann; Schmid, Roland M; Bauer, Stefan; Krug, Anne

    2006-04-15

    Plasmacytoid dendritic cells (PDCs), which produce IFN-alpha in response to autoimmune complexes containing nuclear antigens, are thought to be critically involved in the pathogenesis of systemic lupus erythematosus (SLE). One of the immunostimulatory components of SLE immune complexes (SLE-ICs) is self DNA, which is recognized through Tlr9 in PDCs and B cells. Small nuclear ribonucleoproteins (snRNPs) are another major component of SLE-ICs in 30% to 40% of patients. In this study, we show that murine PDCs are activated by purified U1snRNP/anti-Sm ICs to produce IFN-alpha and proinflammatory cytokines and to up-regulate costimulatory molecules. The induction of IFN-alpha and IL-6 by U1snRNPs in murine bone marrow-derived PDCs required the presence of intact U1RNA and was largely dependent on Tlr7 but independent of Tlr3. Intracellularly delivered isolated U1snRNA and oligoribonucleotides derived from the stem loop regions and the Sm-binding site of U1snRNA efficiently induced IFN-alpha and IL-6 in Flt3L-cultured DCs in a Tlr7-dependent manner. The U1snRNA component of U1snRNP immune complexes, found in patients with SLE, acts as an endogenous "self" ligand for Tlr7 and triggers IFN-alpha and IL-6 production in PDCs. PMID:16368889

  8. An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

    PubMed Central

    Taoka, Masato; Yamauchi, Yoshio; Nobe, Yuko; Masaki, Shunpei; Nakayama, Hiroshi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Isobe, Toshiaki

    2009-01-01

    We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a ∼21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes. PMID:19740761

  9. What Is the Largest Einstein Radius in the Universe?

    SciTech Connect

    Oguri, Masamune; Blandford, Roger D.

    2008-08-05

    The Einstein radius plays a central role in lens studies as it characterizes the strength of gravitational lensing. In particular, the distribution of Einstein radii near the upper cutoff should probe the probability distribution of the largest mass concentrations in the universe. Adopting a triaxial halo model, we compute expected distributions of large Einstein radii. To assess the cosmic variance, we generate a number of Monte-Carlo realizations of all-sky catalogues of massive clusters. We find that the expected largest Einstein radius in the universe is sensitive to parameters characterizing the cosmological model, especially {sigma}{sub s}: for a source redshift of unity, they are 42{sub -7}{sup +9}, 35{sub -6}{sup +8}, and 54{sub -7}{sup +12} arcseconds (errors denote 1{sigma} cosmic variance), assuming best-fit cosmological parameters of the Wilkinson Microwave Anisotropy Probe five-year (WMAP5), three-year (WMAP3) and one-year (WMAP1) data, respectively. These values are broadly consistent with current observations given their incompleteness. The mass of the largest lens cluster can be as small as {approx} 10{sup 15} M{sub {circle_dot}}. For the same source redshift, we expect in all-sky {approx} 35 (WMAP5), {approx} 15 (WMAP3), and {approx} 150 (WMAP1) clusters that have Einstein radii larger than 2000. For a larger source redshift of 7, the largest Einstein radii grow approximately twice as large. While the values of the largest Einstein radii are almost unaffected by the level of the primordial non-Gaussianity currently of interest, the measurement of the abundance of moderately large lens clusters should probe non-Gaussianity competitively with cosmic microwave background experiments, but only if other cosmological parameters are well-measured. These semi-analytic predictions are based on a rather simple representation of clusters, and hence calibrating them with N-body simulations will help to improve the accuracy. We also find that these 'superlens

  10. Numerical simulation of morphodynamic diversity in the World's largest rivers

    NASA Astrophysics Data System (ADS)

    Nicholas, A.

    2012-04-01

    The World's largest rivers share many common properties, including gentle longitudinal bed gradients (~ 0.01-0.1 m per km), high mean annual discharges (~ >10,000 cumecs), and sand-sized bed sediment (D50 ~ 0.1-0.4 mm), yet despite these similarities they are characterised by diverse planform patterns and morphodynamic behaviour (including meandering, braided and anabranching river styles). Recent studies have shown that this diversity cannot be explained using existing channel pattern classification schemes that apply to small rivers. Indeed at present, the causes of morphodynamic diversity in the World's largest rivers remain unclear. Moreover, elucidation of process-form interactions in large rivers is hampered by logistical difficulties involved in field data collection, and by the time period over which satellite imagery is available, which is short given the slow rates of channel change in many large rivers. Numerical models provide a further possible approach for investigating large river morphodynamics. However, although many such models exist, they have generally been developed or applied to simulate either meandering or braided rivers, rather than to investigate a range of channel styles. This paper aims to address this shortcoming using a new numerical simulation model, which is applied to explore the controls on morphodynamic diversity in large sand-bed rivers. This model is based on the 2D shallow water equations with secondary circulation correction, with model components representing total sand transport, suspended transport of silt and clay, bank erosion, vegetation growth and floodplain development. Numerical simulations representing time periods of c. 200 years illustrate how a wide range of channel morphologies, including meandering, braided and anabranching channels, may develop from the same initial conditions and external forcing (valley gradient and discharge regime). These results shed light on the process controls on morphodynamic diversity

  11. Isolation and characterization of the heterogeneous nuclear RNA-ribonucleoprotein complex

    SciTech Connect

    Choi, Y.D.

    1985-01-01

    Exposure of cells to UV light of sufficient intensity brings about crosslinking of RNA to proteins which are in direct contact with it in vivo. The major (/sup 35/S)methionine-labeled proteins which become crosslinked to poly(A)/sup +/hnRNA in HeLa cells are of 120K, 68K, 53K, 43K, 41K, 38K, and 36K (K = kilodaltons). By immunizing mice with UV crosslinked complexes two monoclonal antibodies (2B12 and 4F4) against the C proteins (41K and 43K) and one (3G6) against the 120K protein of the hnRNP complex were obtained. Immunofluorescence microscopy demonstrates that the C proteins and 120K are segregated to the nucleus and are not associated with nucleoli or chromatin. The two C proteins are highly related to each other antigenically. Monoclonal antibody 4F4 identifies the C proteins of the hnRNP complex in widely divergent species from human to lizard. The C proteins are phosphorylated and are in contact with hnRNA in vivo. The hnRNP complex was isolated from vertebrate cell nuclei by immunoprecipitation with these monoclonal antibodies. This complex contains proteins and hnRNA of up to approx.10 kb. The major steady state labeled (/sup 35/S)methionine labeled proteins of the isolated complex from HeLa cells are of 34K, 36K, 36K (A1 and A2), 37K, 38K (B1 and B2), 41K, 43K (C1 and C2) and doublets at 68K and at 120K. These proteins are organized into a 30S particle. Large hnRNP complexes are composed of multiples of 30S particles which are connected by highly nuclease sensitive stretches of hnRNA. It it concluded that the hnRNP structure is an integral component of the mRNA formation pathway in the eukaryotic cell.

  12. Testing a Parachute for Mars in World's Largest Wind Tunnel

    NASA Technical Reports Server (NTRS)

    2007-01-01

    The team developing the landing system for NASA's Mars Science Laboratory tested the deployment of an early parachute design in mid-October 2007 inside the world's largest wind tunnel, at NASA Ames Research Center, Moffett Field, California.

    In this image, two engineers are dwarfed by the parachute, which holds more air than a 280-square-meter (3,000-square-foot) house and is designed to survive loads in excess of 36,000 kilograms (80,000 pounds).

    The parachute, built by Pioneer Aerospace, South Windsor, Connecticut, has 80 suspension lines, measures more than 50 meters (165 feet) in length, and opens to a diameter of nearly 17 meters (55 feet). It is the largest disk-gap-band parachute ever built and is shown here inflated in the test section with only about 3.8 meters (12.5 feet) of clearance to both the floor and ceiling.

    The wind tunnel, which is 24 meters (80 feet) tall and 37 meters (120 feet) wide and big enough to house a Boeing 737, is part of the National Full-Scale Aerodynamics Complex, operated by the U.S. Air Force, Arnold Engineering Development Center.

    NASA's Jet Propulsion Laboratory, Pasadena, California, is building and testing the Mars Science Laboratory spacecraft for launch in 2009. The mission will land a roving analytical laboratory on the surface of Mars in 2010. JPL is a division of the California Institute of Technology.

  13. Historically largest geomagnetic sudden commencement (SC) since 1868

    NASA Astrophysics Data System (ADS)

    Araki, Tohru

    2014-12-01

    Being stimulated by the previously reported large amplitude (202 nT at Kakioka) geomagnetic sudden commencement (SC) on 24 March 1991, we searched larger amplitude SCs in the past. We tried to collect old magnetograms and used the list of SC observed at Kakioka (27.5° gm.lat.) for the period 1924 to 2013 and Colaba (10.5°)-Alibag (10.3°) for 1868 to 1967. We found that the largest amplitude SC occurred on 24 March (the same day as 1991 SC), 1940. The H-component amplitude is larger than 273 nT at Kakioka and 310 nT at Alibag. We could also obtain the copy of the magnetogram of Cape Town (-33.3°) which shows 164 nT amplitude. The statistical analysis shows that the occurrence rate of SCs is less than 5% for amplitude larger than 50 nT and less than 1% for amplitude larger than 100 nT at both Kakioka and Alibag. Large amplitude SCs tend to occur during the declining phase of the solar activity. Finally, we discussed the possible increase of the dynamic pressure associated with the interplanetary shock causing the largest SC.

  14. Heterogeneous Nuclear Ribonucleoprotein C1/C2 Controls the Metastatic Potential of Glioblastoma by Regulating PDCD4

    PubMed Central

    Park, Young Mi; Hwang, Su Jin; Masuda, Kiyoshi; Choi, Kyung-Min; Jeong, Mi-Ran; Nam, Do-Hyun

    2012-01-01

    MicroRNAs (miRNAs) have been implicated in the pathogenesis and progression of brain tumors. miR-21 is one of the most highly overexpressed miRNAs in glioblastoma multiforme (GBM), and its level of expression correlates with the tumor grade. Programmed cell death 4 (PDCD4) is a well-known miR-21 target and is frequently downregulated in glioblastomas in accordance with increased miR-21 expression. Downregulation of miR-21 or overexpression of PDCD4 can inhibit metastasis. Here, we investigate the role of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) in the metastatic potential of the glioblastoma cell line T98G. hnRNPC bound directly to primary miR-21 (pri-miR-21) and promoted miR-21 expression in T98G cells. Silencing of hnRNPC lowered miR-21 levels, in turn increasing the expression of PDCD4, suppressing Akt and p70S6K activation, and inhibiting migratory and invasive activities. Silencing of hnRNPC reduced cell proliferation and enhanced etoposide-induced apoptosis. In support of a role for hnRNPC in the invasiveness of GBM, highly aggressive U87MG cells showed higher hnRNPC expression levels and hnRNPC abundance in tissue arrays and also showed elevated levels as a function of brain tumor grade. Taken together, our data indicate that hnRNPC controls the aggressiveness of GBM cells through the regulation of PDCD4, underscoring the potential usefulness of hnRNPC as a prognostic and therapeutic marker of GBM. PMID:22907752

  15. A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons.

    PubMed

    Lico, D T P; Lopes, G S; Brusco, J; Rosa, J C; Gould, R M; De Giorgis, J A; Larson, R E

    2015-08-01

    The presence of mRNAs in synaptic terminals and their regulated translation are important factors in neuronal communication and plasticity. Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are involved in the translocation, stability, and subcellular localization of mRNA and the regulation of its translation. Defects in these processes and mutations in components of the hnRNP complexes have been related to the formation of cytoplasmic inclusion bodies and neurodegenerative diseases. Despite much data on mRNA localization and evidence for protein synthesis, as well as the presence of translation machinery, in axons and presynaptic terminals, the identity of RNA-binding proteins involved in RNA transport and function in presynaptic regions is lacking. We previously characterized a strongly basic RNA-binding protein (p65), member of the hnRNPA/B subfamily, in squid presynaptic terminals. Intriguingly, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), p65 migrated as a 65-kDa protein, whereas members of the hnRNPA/B family typically have molecular masses ranging from 35 to 42kDa. In this report we present further biochemical and molecular characterization that shows endogenous p65 to be an SDS-stable dimer composed of ∼37-kDa hnRNPA/B-like subunits. We cloned and expressed a recombinant protein corresponding to squid hnRNPA/B-like protein and showed its propensity to aggregate and form SDS-stable dimers in vitro. Our data suggest that this unique hnRNPA/B-like protein co-localizes with synaptic vesicle protein 2 and RNA-binding protein ELAV and thus may serve as a link between local mRNA processing and presynaptic function and regulation. PMID:26012490

  16. Cyclophilin E Functions as a Negative Regulator to Influenza Virus Replication by Impairing the Formation of the Viral Ribonucleoprotein Complex

    PubMed Central

    Wang, Zengfu; Liu, Xiaoling; Zhao, Zhendong; Xu, Chongfeng; Zhang, Ke; Chen, Caiwei; Sun, Lei; Gao, George F.; Ye, Xin; Liu, Wenjun

    2011-01-01

    Background The nucleoprotein (NP) of influenza A virus is a multifunctional protein that plays a critical role in the replication and transcription of the viral genome. Therefore, examining host factors that interact with NP may shed light on the mechanism of host restriction barriers and the tissue tropism of influenza A virus. Here, Cyclophilin E (CypE), a member of the peptidyl-propyl cis-trans isomerase (PPIase) family, was found to bind to NP and inhibit viral replication and transcription. Methodology/Principal Findings In the present study, CypE was found to interact with NP but not with the other components of the viral ribonucleoprotein complex (vRNP): PB1, PB2, and PA. Mutagenesis data revealed that the CypE domain comprised of residues 137–186 is responsible for its binding to NP. Functional analysis results indicated that CypE is a negative regulator in the influenza virus life cycle. Furthermore, knock-down of CypE resulted in increased levels of three types of viral RNA, suggesting that CypE negatively affects viral replication and transcription. Moreover, up-regulation of CypE inhibited the activity of influenza viral polymerase. We determined that the molecular mechanism by which CypE negatively regulates influenza virus replication and transcription is by interfering with NP self-association and the NP-PB1 and NP-PB2 interactions. Conclusions/Significance CypE is a host restriction factor that inhibits the functions of NP, as well as viral replication and transcription, by impairing the formation of the vRNP. The data presented here will help us to better understand the molecular mechanisms of host restriction barriers, host adaptation, and tissue tropism of influenza A virus. PMID:21887220

  17. Heterogeneous nuclear ribonucleoprotein K upregulates the kinetochore complex component NUF2 and promotes the tumorigenicity of colon cancer cells

    SciTech Connect

    Sugimasa, Hironobu; Taniue, Kenzui; Kurimoto, Akiko; Takeda, Yasuko; Kawasaki, Yoshihiro; Akiyama, Tetsu

    2015-03-27

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein involved in transcription, mRNA splicing, mRNA stabilization and translation. Although hnRNP K has been suggested to play a role in the development of many cancers, its molecular function in colorectal cancer has remained elusive. Here we show that hnRNP K plays an important role in the mitotic process in HCT116 colon cancer cells. Furthermore, we demonstrate that hnRNP K directly transactivates the NUF2 gene, the product of which is a component of the NDC80 kinetochore complex and which is known to be critical for a stable spindle microtubule-kinetochore attachment. In addition, knockdown of both hnRNP K and NUF2 caused failure in metaphase chromosome alignment and drastic decrease in the growth of colon cancer cells. These results suggest that the hnRNP K-NUF2 axis is important for the mitotic process and proliferation of colon cancer cells and that this axis could be a target for the therapy of colon cancer. - Highlights: • hnRNP K is required for the tumorigenicity of colon cancer cells. • hnRNP K binds to the promoter region of NUF2 and activates its transcription. • NUF2 expression is correlated with hnRNP K expression in colorectal cancer tissue. • hnRNP K and NUF2 are required for metaphase chromosome alignment. • The hnRNP K-NUF2 axis is important for the proliferation of colon cancer cells.

  18. Earth's Largest Meteorite Impact Craters discovered in South America?

    NASA Astrophysics Data System (ADS)

    Kellndorfer, J. M.; Schmidt-Falkenberg, H.

    2014-12-01

    Novel analysis of high resolution InSAR-based digital elevation data from the year 2001 Shuttle Radar Topography Mission combined with a recently produced dataset of pan-tropical vegetation height from ALOS-1 SAR and IceSAT/GLAS Lidar estimates led to the quasi-bald-Earth discovery of four sizable near-perfect circle arcs in South America under dense tropical forests ranging in length from 216 km to 441 km. Terrain elevation profiles of cross-sections across the arcs show a distinct vertical rising and falling in elevations of hundreds of meters over a horizontal distance of tens of kilometers. It is hypothesized that these sizable arcs and associated rim-like topographic terrain features are remnants of huge meteorite impact craters with diameters ranging from 770 km to 1,310 km, thus forming potentially the largest known impact carter structures discovered on Earth today. The potential impact crater rim structures are located north of the eastern Amazon River, in the coastal region of Recife and Natal, and in the Brazilian, Bolivian and Paraguayan border region encompassing the Pantanal. Elevation profiles, hillshades and gray-shaded elevation maps were produced to support the geomorphologic analysis. It is also speculated whether in three of the four potential impact craters, central uplift domes or peaks, which are typical for complex impact crater structures can be identified. The worlds largest iron ore mining area of Carajás in Para, Brazil, falls exactly in the center of the largest hypothesized circular impact crater showing topographic elevations similar to the rim structure discovered 655 km to the north-north-west. Based on the topographic/geomorphologic driven hypothesis, geologic exploration of these topographic features is needed to test whether indeed meteorite impact craters could be verified, what the more exact ellipsoidal shapes of the potential impact craters might be, and to determine when during geologic times the impacts would have taken

  19. The largest Silurian vertebrate and its palaeoecological implications

    PubMed Central

    Choo, Brian; Zhu, Min; Zhao, Wenjin; Jia, Liaotao; Zhu, You'an

    2014-01-01

    An apparent absence of Silurian fishes more than half-a-metre in length has been viewed as evidence that gnathostomes were restricted in size and diversity prior to the Devonian. Here we describe the largest pre-Devonian vertebrate (Megamastax amblyodus gen. et sp. nov.), a predatory marine osteichthyan from the Silurian Kuanti Formation (late Ludlow, ~423 million years ago) of Yunnan, China, with an estimated length of about 1 meter. The unusual dentition of the new form suggests a durophagous diet which, combined with its large size, indicates a considerable degree of trophic specialisation among early osteichthyans. The lack of large Silurian vertebrates has recently been used as constraint in palaeoatmospheric modelling, with purported lower oxygen levels imposing a physiological size limit. Regardless of the exact causal relationship between oxygen availability and evolutionary success, this finding refutes the assumption that pre-Emsian vertebrates were restricted to small body sizes. PMID:24921626

  20. The depths of the largest impact craters on Venus

    NASA Technical Reports Server (NTRS)

    Ivanov, B. A.; Ford, P. G.

    1993-01-01

    The largest impact craters on Venus may be used as evidence of various geological processes within the Venusian crust. We are continuing to construct a data base for the further investigation of large craters on Venus (LCV). We hope to find evidence of crater relaxation that might constrain the thickness and thermal gradient of the crust, as was proposed in an earlier work. The current work concentrates on 27 impact craters with diameters (d) larger than 70 km, i.e., large enough that the footprint of the Magellan altimeter has a good chance of sampling the true crater bottom. All altimeter echoes from points located within (d/2)+70 km from the crater center have been inspected.

  1. The World's Largest Real Time Magnetometer Array: MAGDAS

    NASA Astrophysics Data System (ADS)

    Maeda, G.; Yumoto, K.; Abe, S.; Uozumi, T.

    2009-12-01

    This poster briefly out lines the current situation of the world's largest real time magnetometer array: MAGDAS. This array was conceived by Professor K. Yumoto of the Space Environment Research Center (SERC) based at Kyushu University in Japan. The first phase of MAGDAS deployment coincided with IHY (Years 2005-2009) and about 50 units were installed all over the world. The second phase of MAGDAS deployment is set to occur during ISWI (Intl. Space Weather Initiative, Years 2010-2012) using a new set of 50 magnetometers. MAGDAS stations are concentrated in three chains: (1) the 210 deg. MM chain through Asia, (2) the 96 deg. MM chain through Africa, and (3) the Dip Equator Chain. Data from MAGDAS is available to the scientific community but some conditions are attached to the data. We explain those conditions.

  2. Basal resistance for three of the largest Greenland outlet glaciers

    NASA Astrophysics Data System (ADS)

    Shapero, Daniel R.; Joughin, Ian R.; Poinar, Kristin; Morlighem, Mathieu; Gillet-Chaulet, Fabien

    2016-01-01

    Resistance at the ice-bed interface provides a strong control on the response of ice streams and outlet glaciers to external forcing, yet it is not observable by remote sensing. We used inverse methods constrained by satellite observations to infer the basal resistance to flow underneath three of the Greenland Ice Sheet's largest outlet glaciers. In regions of fast ice flow and high (>250 kPa) driving stresses, ice is often assumed to flow over a strong bed. We found, however, that the beds of these three glaciers provide almost no resistance under the fast-flowing trunk. Instead, resistance to flow is provided by the lateral margins and stronger beds underlying slower-moving ice upstream. Additionally, we found isolated patches of high basal resistivity within the predominantly weak beds. Because these small-scale (<1 ice thickness) features may be artifacts of overfitting our solution to measurement errors, we tested their robustness to different degrees of regularization.

  3. Water, Sanitation, and Hygiene at the World's Largest Mass Gathering.

    PubMed

    Vortmann, Michael; Balsari, Satchit; Holman, Susan R; Greenough, P Gregg

    2015-02-01

    The 2013 Kumbh Mela, a Hindu religious festival and the largest human gathering on earth, drew an estimated 120 million pilgrims to bathe at the holy confluence of the Ganga (Ganges) and Yamuna rivers. To accommodate the massive numbers, the Indian government constructed a temporary city on the flood plains of the two rivers and provided it with roads, electricity, water and sanitation facilities, police stations, and a tiered healthcare system. This phenomenal operation and its impacts have gone largely undocumented. To address this gap, the authors undertook an evaluation and systematic monitoring initiative to study preparedness and response to public health emergencies at the event. This paper describes the water, sanitation, and hygiene components, with particular emphasis on preventive and mitigation strategies; the capacity for surveillance and response to diarrheal disease outbreaks; and the implications of lessons learned for other mass gatherings. PMID:25783442

  4. Correlates of species richness in the largest Neotropical amphibian radiation

    PubMed Central

    Gonzalez-Voyer, A; Padial, J M; Castroviejo-Fisher, S; De La Riva, I; Vilà, C

    2011-01-01

    Although tropical environments are often considered biodiversity hotspots, it is precisely in such environments where least is known about the factors that drive species richness. Here, we use phylogenetic comparative analyses to study correlates of species richness for the largest Neotropical amphibian radiation: New World direct-developing frogs. Clade-age and species richness were nonsignficantly, negatively correlated, suggesting that clade age alone does not explain among-clade variation in species richness. A combination of ecological and morphological traits explained 65% of the variance in species richness. A more vascularized ventral skin, the ability to colonize high-altitude ranges, encompassing a large variety of vegetation types, correlated significantly with species richness, whereas larger body size was marginally correlated with species richness. Hence, whereas high-altitude ranges play a role in shaping clade diversity in the Neotropics, intrinsic factors, such as skin structures and possibly body size, might ultimately determine which clades are more speciose than others. PMID:21401771

  5. On the macroseismic magnitudes of the largest Italian earthquakes

    NASA Astrophysics Data System (ADS)

    Tinti, S.; Vittori, T.; Mulargia, F.

    1987-07-01

    The macroseismic magnitudes MT of the largest Italian earthquakes ( I0 ⩾ VIII, MCS) have been computed by using the intensity magnitude relationships recently assessed by the authors (1986) for the Italian region. The Progetto Finalizzato Geodinamica (PFG) catalog of the Italian earthquakes, covering the period 1000-1980 (Postpischl, 1985) is the source data base and is reproduced in the Appendix: here the estimated values of MT are given side by side with the catalog macroseismic magnitudes MK i.e. the magnitudes computed according to the Karnik laws (Karnik, 1969). The one-sigma errors Δ MT are also given for each earthquake. The basic aim of the paper is to provide a handy and useful tool to researchers involved in seismicity and seismic-risk studies on Italian territory.

  6. 1. Photocopy of undated wash drawing. The largest building is ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. Photocopy of undated wash drawing. The largest building is the Hotel Williams. Next to it is the Williams House (Williams Hotel Annex), HABS No. MI-258 A. Below them are the log cabins built by the American Fur Company (from left to right): Log Building No. 1 (MI-258 C), Log Building No. 2 (MI-258 D), Log Building No. 3 (not documented), Log Building No. 4 (MI-258 E), and Log Building No. 5 (MI-258 F). (Williams Log House, MI-258 B, and Log Building No. 8, MI-258 G, are not shown). The drawing is in the collection of the Michigan Historical Commission. Although it does not have a date, it was painted in the early 20th century after the main building was constructed. - Hotel Williams, Murray Bay, Munising, Alger County, MI

  7. Collapse of the world’s largest herbivores

    PubMed Central

    Ripple, William J.; Newsome, Thomas M.; Wolf, Christopher; Dirzo, Rodolfo; Everatt, Kristoffer T.; Galetti, Mauro; Hayward, Matt W.; Kerley, Graham I. H.; Levi, Taal; Lindsey, Peter A.; Macdonald, David W.; Malhi, Yadvinder; Painter, Luke E.; Sandom, Christopher J.; Terborgh, John; Van Valkenburgh, Blaire

    2015-01-01

    Large wild herbivores are crucial to ecosystems and human societies. We highlight the 74 largest terrestrial herbivore species on Earth (body mass ≥100 kg), the threats they face, their important and often overlooked ecosystem effects, and the conservation efforts needed to save them and their predators from extinction. Large herbivores are generally facing dramatic population declines and range contractions, such that ~60% are threatened with extinction. Nearly all threatened species are in developing countries, where major threats include hunting, land-use change, and resource depression by livestock. Loss of large herbivores can have cascading effects on other species including large carnivores, scavengers, mesoherbivores, small mammals, and ecological processes involving vegetation, hydrology, nutrient cycling, and fire regimes. The rate of large herbivore decline suggests that ever-larger swaths of the world will soon lack many of the vital ecological services these animals provide, resulting in enormous ecological and social costs. PMID:26601172

  8. Opportunities for biodiversity gains under the world's largest reforestation programme.

    PubMed

    Hua, Fangyuan; Wang, Xiaoyang; Zheng, Xinlei; Fisher, Brendan; Wang, Lin; Zhu, Jianguo; Tang, Ya; Yu, Douglas W; Wilcove, David S

    2016-01-01

    Reforestation is a critical means of addressing the environmental and social problems of deforestation. China's Grain-for-Green Program (GFGP) is the world's largest reforestation scheme. Here we provide the first nationwide assessment of the tree composition of GFGP forests and the first combined ecological and economic study aimed at understanding GFGP's biodiversity implications. Across China, GFGP forests are overwhelmingly monocultures or compositionally simple mixed forests. Focusing on birds and bees in Sichuan Province, we find that GFGP reforestation results in modest gains (via mixed forest) and losses (via monocultures) of bird diversity, along with major losses of bee diversity. Moreover, all current modes of GFGP reforestation fall short of restoring biodiversity to levels approximating native forests. However, even within existing modes of reforestation, GFGP can achieve greater biodiversity gains by promoting mixed forests over monocultures; doing so is unlikely to entail major opportunity costs or pose unforeseen economic risks to households. PMID:27598524

  9. The 260: The Largest Solid Rocket Motor Ever Tested

    NASA Technical Reports Server (NTRS)

    Crimmins, P.; Cousineau, M.; Rogers, C.; Shell, V.

    1999-01-01

    Aerojet in the mid 1960s, under contract to NASA, built and static hot fire tested the largest solid rocket motor (SRM) in history for the purpose of demonstrating the feasibility of utilizing large SRMs for space exploration. This program successfully fabricated two high strength steel chambers, loaded each with approximately 1,68 million pounds of propellant, and static test fired these giants with their nozzles up from an underground silo located adjacent to the Florida everglades. Maximum thrust and total impulse in excess of 5,000,000 lbf and 3,470,000,000 lbf-sec were achieved. Flames from the second firing, conducted at night, were seen over eighty miles away. For comparative purposes: the thrust developed was nearly 100 times that of a Minuteman III second stage and the 260 in.-dia cross-section was over 3 times that of the Space Shuttle SRM.

  10. Supergranulation as the Sun's largest buoyantly driven mode of convection

    NASA Astrophysics Data System (ADS)

    Cossette, Jean-Francois; Rast, Mark

    2016-05-01

    Solar supergranulation has been characterized as horizontally divergent flow motions having a typical scale of 32 Mm using Doppler imaging, granule tracking and helioseismology. Unlike granules, the size of which is comparable to both the thickness of the radiative boundary layer and local scale height at the photosphere, supergranules do not appear to correspond to any particular length scale of the flow. Possible explanations ranging from convection theories involving Helium ionization to spatial correlation or self-organization of granular flows have been proposed as physical mechanisms to explain solar supergranulation. However, its existence remains largely a mystery. Remarkably, horizontal velocity power spectra obtained from Doppler imaging and correlation tracking of flow features at the solar surface reveal the presence of peaks corresponding to granular and supergranular scales, followed by a monotonic decrease in power at scales larger than supergranulation, which suggests that large-scale modes in the deep layers of the convection zone may be suppressed. Using 3D anelastic simulations of solar convection we investigate whether supergranulation may reflect the largest buoyantly driven mode of convection inside the Sun. Results show that the amount of kinetic energy contained in the largest flow scales relative to that associated with supergranular motions is a function of the depth of the transition from a convectively unstable to convectively stable mean stratification inside the simulation. This suggests that the observed monotonic decrease in power at scales larger than supergranulation may be explained by rapid cooling in the subphotospheric layers and an essentially isentropic solar interior, wherein convective driving is effectively suppressed.

  11. Largest Aftershocks of Megathrust Earthquakes in the World

    NASA Astrophysics Data System (ADS)

    Koyama, J.; Tsuzuki, M.

    2012-12-01

    The 2011 Tohoku-oki megathrust earthquake of Mw9.0 induced the earthquake activity in high level all over Japan. It included not only earthquakes near active faults but also volcanic earthquakes. Although we have observed tens of thousands of aftershocks, yet we do not know which is the largest aftershock of the 2011 megathrust. There occurred several megathrust earthquakes worldwide in the last one hundred years, which are almost the same size or larger than the 2011 megathrust. We have studied their largest aftershocks based on our new hypothesis of along-dip double segmentation (ADDS) and along-strike single segmentation (ASSS). ADDS in the Tohoku-oki region along the Japan trench is characterized by the apparent absence of earthquakes in the trench-ward segments as opposed to the Japan Island-ward segments that have repeated small earthquakes of up to Mw8 class. In contrast, the 1960 Chile and the 2010 Maule megathrusts are characterized by ASSS with the weak seismic activity before the main event everywhere in the subduction zone. The difference between these two types of seismic segmentations would be that strongly coupled areas of trench-ward segments give rise to ADDS, whereas almost 100% coupled areas of shallow-parts of subduction zones give rise to ASSS. In other words, the phenomenon of a seismic gap can be identified for an ASSS megathrust, where as a doughnut pattern of seismic activity appears prior to a main ADDS event. In summary, most of the largest aftershocks of ADDS megathrusts are earthquakes of outer-rise(outer trench-slope) normal faultings, where there occur two types, dip-slip and strike-slip, depending on the structure of subducting oceanic plates. The 1933 Sanriku-oki Mw8.6 (the 1896 Meiji-Sanriku M~8.5) and the 2011 Tohoku-oki Mw7.7 (the 2011 Tohoku-oki Mw9.0) are the former and the 1987 Off Alaska Mw7.8 (the 1964 Alaska Mw9.2) and the 2012 Sumatra Mw8.6 (the 2004 Sumatra-Andaman Mw9.3) are the latter. Those of ASSS megathrusts occurred

  12. Urban aerosol pollution assessment in Russian largest cities

    NASA Astrophysics Data System (ADS)

    Malinina, Elizaveta; Chubarova, Natalia; Sviridenkov, Mikhail

    2015-04-01

    We assessed the aerosol pollution in 51 Russian largest cities, which are located in different climatic zones with various level of urban pollution using the aerosol optical thickness (AOT) at 550 nm from MODIS data with a spatial resolution 1°×1°during the warm period of 2000-2013. For better understanding of MODIS radiometers quality of AOT data over Russia the comparisons between AERONET data and MODIS data were fulfilled. They showed a good quality of the MODIS data and its possibility to detect an urban pollution from space. As a characteristic of urban aerosol pollution we used the difference between AOT in the city and the background value of AOT in the nearby region. The research showed, that urban aerosol pollution in Russian cities varied from 0.01 to 0.08. Besides, more precise assessment of urban aerosol pollution in Moscow was done basing on the AERONET network data in two sites at Moscow University Meteorological Observatory (MO) and at the Zvenigorod Scientific Station (ZSS). For the assessment the simultaneous measurements at these two sites during the period from September 2006 till July 2013 were taken. The average AOT difference in the visible spectrum (wavelength: 500 nm) between MO and ZSS, which could be used as pollution characteristic, is about 0.02. These results are similar to similar research, which had been made earlier for a shorter period. The difference between AOT at these two sites is the largest in winter and is about 0.03. In order to assess the dominating aerosol components in different cities we used the data of Russian network on emissions and concentrations of the main pollutants over the 1988-2011 period. For each city we evaluated the ratio of sulfur dioxide to nitrogen dioxide emissions and its trends to assess the dominating aerosol properties and temporal variability. As a result, the radiative forcing of urban aerosol pollution for different cities was evaluated. Depending on the location, the size and the level of

  13. Spectral properties of the largest asteroids associated with Taurid Complex

    NASA Astrophysics Data System (ADS)

    Popescu, M.; Birlan, M.; Nedelcu, D. A.; Vaubaillon, J.; Cristescu, C. P.

    2014-12-01

    Context. The Taurid Complex is a massive stream of material in the inner part of the Solar System. It contains objects spanning the range of 10-6-103 m, considered by some authors to have a common cometary origin. The asteroids belonging to Taurid Complex are on Apollo type orbit, with most of them being flagged as potentially hazardous asteroids. In this context, understanding the nature and the origin of this asteroidal population is not only of scientific interest but also of practical importance. Aims: We aim to investigate the surface mineralogy of the asteroids associated with Taurid Complex using visible and near-infrared spectral data. Compositional linking between these asteroids and meteorites can be derived based on the obtained spectra. Methods: We obtained spectra of six of the largest asteroids (2201, 4183, 4486, 5143, 6063, and 269690) associated with Taurid complex. The observations were made with the IRTF telescope equipped with the spectro-imager SpeX. Their taxonomic classification is made using Bus-DeMeo taxonomy. The asteroid spectra are compared with the meteorite spectra from the Relab database. Mineralogical models were applied to determine their surface composition. All the spectral analysis is made in the context of the already published physical data. Results: Five of the objects studied in this paper present spectral characteristics similar to the S taxonomic complex. The spectra of ordinary chondrites (spanning H, L, and LL subtypes) are the best matches for these asteroid spectra. The asteroid (269690) 1996 RG3 presents a flat featureless spectrum which could be associated to a primitive C-type object. The increased reflectance above 2.1 microns constrains its geometrical albedo to a value around 0.03. Conclusions: While there is an important dynamical grouping among the Taurid Complex asteroids, the spectral data of the largest objects do not support a common cometary origin. Furthermore, there are significant variations between the

  14. Largest US oil and gas fields, August 1993

    SciTech Connect

    Not Available

    1993-08-06

    The Largest US Oil and Gas Fields is a technical report and part of an Energy Information Administration (EIA) series presenting distributions of US crude oil and natural gas resources, developed using field-level data collected by EIA`s annual survey of oil and gas proved reserves. The series` objective is to provide useful information beyond that routinely presented in the EIA annual report on crude oil and natural gas reserves. These special reports also will provide oil and gas resource analysts with a fuller understanding of the nature of US crude oil and natural gas occurrence, both at the macro level and with respect to the specific subjects addressed. The series` approach is to integrate EIA`s crude oil and natural gas survey data with related data obtained from other authoritative sources, and then to present illustrations and analyses of interest to a broad spectrum of energy information users ranging from the general public to oil and gas industry personnel.

  15. Claim of largest flood on record proves false

    NASA Astrophysics Data System (ADS)

    Sheffer, N. A.; Enzel, Y.; Waldmann, N.; Grodek, T.; Benito, G.

    A study of paleofloods in the Gardon River in southern France reveals the occurrence of past extreme floods that were larger than any observed historically. From 8 to 9 September 2002, during the course of the study by a complete coincidence, an extreme flood claimed the lives of 21 people and caused millions of dollars worth of damage to the towns and villages along the river. This flood was larger in magnitude than any flood on record, according to gaged data since 1890.An autumn storm, which is typical of this region, struck with immense force. The rain cell migrated from the lower reaches of the basin on the evening of 8 September to the upper parts of the basin, producing 680 mm of rain in 20 hours.The flood's peak discharge is preliminary estimated at 6000 m3 s-1. This flood is now considered by the media and professionals to be “The largest flood on record.” However, our research proves otherwise.

  16. World's Largest Gold Crystal Studied at Los Alamos

    ScienceCinema

    Vogel, Sven; Nakotte, Heinz

    2015-01-07

    When geologist John Rakovan needed better tools to investigate whether a dazzling 217.78-gram piece of gold was in fact the world's largest single-crystal specimen - a distinguishing factor that would not only drastically increase its market value but also provide a unique research opportunity - he traveled to Los Alamos National Laboratory's Lujan Neutron Scattering Center to peer deep inside the mineral using neutron diffractometry. Neutrons, different from other probes such as X-rays and electrons, are able to penetrate many centimeters deep into most materials. Revealing the inner structure of a crystal without destroying the sample - imperative, as this one is worth an estimated $1.5 million - would allow Rakovan and Lujan Center collaborators Sven Vogel and Heinz Nakotte to prove that this exquisite nugget, which seemed almost too perfect and too big to be real, was a single crystal and hence a creation of nature. Its owner, who lives in the United States, provided the samples to Rakovan to assess the crystallinity of four specimens, all of which had been found decades ago in Venezuela.

  17. Holocene dynamics of the Arctic's largest ice shelf

    PubMed Central

    Antoniades, Dermot; Francus, Pierre; Pienitz, Reinhard; St-Onge, Guillaume; Vincent, Warwick F.

    2011-01-01

    Ice shelves in the Arctic lost more than 90% of their total surface area during the 20th century and are continuing to disintegrate rapidly. The significance of these changes, however, is obscured by the poorly constrained ontogeny of Arctic ice shelves. Here we use the sedimentary record behind the largest remaining ice shelf in the Arctic, the Ward Hunt Ice Shelf (Ellesmere Island, Canada), to establish a long-term context in which to evaluate recent ice-shelf deterioration. Multiproxy analysis of sediment cores revealed pronounced biological and geochemical changes in Disraeli Fiord in response to the formation of the Ward Hunt Ice Shelf and its fluctuations through time. Our results show that the ice shelf was absent during the early Holocene and formed 4,000 years ago in response to climate cooling. Paleoecological data then indicate that the Ward Hunt Ice Shelf remained stable for almost three millennia before a major fracturing event that occurred ∼1,400 years ago. After reformation ∼800 years ago, freshwater was a constant feature of Disraeli Fiord until the catastrophic drainage of its epishelf lake in the early 21st century. PMID:22025693

  18. Characterization of the largest effector gene cluster of Ustilago maydis.

    PubMed

    Brefort, Thomas; Tanaka, Shigeyuki; Neidig, Nina; Doehlemann, Gunther; Vincon, Volker; Kahmann, Regine

    2014-07-01

    In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function. PMID:24992561

  19. [A Standing Balance Evaluation Method Based on Largest Lyapunov Exponent].

    PubMed

    Liu, Kun; Wang, Hongrui; Xiao, Jinzhuang; Zhao, Qing

    2015-12-01

    In order to evaluate the ability of human standing balance scientifically, we in this study proposed a new evaluation method based on the chaos nonlinear analysis theory. In this method, a sinusoidal acceleration stimulus in forward/backward direction was forced under the subjects' feet, which was supplied by a motion platform. In addition, three acceleration sensors, which were fixed to the shoulder, hip and knee of each subject, were applied to capture the balance adjustment dynamic data. Through reconstructing the system phase space, we calculated the largest Lyapunov exponent (LLE) of the dynamic data of subjects' different segments, then used the sum of the squares of the difference between each LLE (SSDLLE) as the balance capabilities evaluation index. Finally, 20 subjects' indexes were calculated, and compared with evaluation results of existing methods. The results showed that the SSDLLE were more in line with the subjects' performance during the experiment, and it could measure the body's balance ability to some extent. Moreover, the results also illustrated that balance level was determined by the coordinate ability of various joints, and there might be more balance control strategy in the process of maintaining balance. PMID:27079089

  20. World's Largest Gold Crystal Studied at Los Alamos

    SciTech Connect

    Vogel, Sven; Nakotte, Heinz

    2014-04-03

    When geologist John Rakovan needed better tools to investigate whether a dazzling 217.78-gram piece of gold was in fact the world's largest single-crystal specimen - a distinguishing factor that would not only drastically increase its market value but also provide a unique research opportunity - he traveled to Los Alamos National Laboratory's Lujan Neutron Scattering Center to peer deep inside the mineral using neutron diffractometry. Neutrons, different from other probes such as X-rays and electrons, are able to penetrate many centimeters deep into most materials. Revealing the inner structure of a crystal without destroying the sample - imperative, as this one is worth an estimated $1.5 million - would allow Rakovan and Lujan Center collaborators Sven Vogel and Heinz Nakotte to prove that this exquisite nugget, which seemed almost too perfect and too big to be real, was a single crystal and hence a creation of nature. Its owner, who lives in the United States, provided the samples to Rakovan to assess the crystallinity of four specimens, all of which had been found decades ago in Venezuela.

  1. Cosmology on the Largest Scales with the SKA

    NASA Astrophysics Data System (ADS)

    Camera, S.; Raccanelli, A.; Bull, P.; Bertacca, D.; Chen, X.; Ferreira, P.; Kunz, M.; Maartens, R.; Mao, Y.; Santos, M.; Shapiro, P. R.; Viel, M.; Xu, Y.

    2015-04-01

    The study of the Universe on ultra-large scales is one of the major science cases for the Square Kilometre Array (SKA). The SKA will be able to probe a vast volume of the cosmos, thus representing a unique instrument, amongst next-generation cosmological experiments, for scrutinising the Universe's properties on the largest cosmic scales. Probing cosmic structures on extremely large scales will have many advantages. For instance, the growth of perturbations is well understood for those modes, since it falls fully within the linear regime. Also, such scales are unaffected by the poorly understood feedback of baryonic physics. On ultra-large cosmic scales, two key effects become significant: primordial non-Gaussianity and relativistic corrections to cosmological observables. Moreover, if late-time acceleration is driven not by dark energy but by modifications to general relativity, then such modifications should become apparent near and above the horizon scale. As a result, the SKA is forecast to deliver transformational constraints on non-Gaussianity and to probe gravity on super-horizon scales for the first time.

  2. Four corners: The largest US methane anomaly viewed from space

    NASA Astrophysics Data System (ADS)

    Kort, Eric A.; Frankenberg, Christian; Costigan, Keeley R.; Lindenmaier, Rodica; Dubey, Manvendra K.; Wunch, Debra

    2014-10-01

    Methane (CH4) is a potent greenhouse gas and ozone precursor. Quantifying methane emissions is critical for projecting and mitigating changes to climate and air quality. Here we present CH4 observations made from space combined with Earth-based remote sensing column measurements. Results indicate the largest anomalous CH4 levels viewable from space over the conterminous U.S. are located at the Four Corners region in the Southwest U.S. Emissions exceeding inventory estimates, totaling 0.59 Tg CH4/yr [0.50-0.67; 2σ], are necessary to bring high-resolution simulations and observations into agreement. This underestimated source approaches 10% of the EPA estimate of total U.S. CH4 emissions from natural gas. The persistence of this CH4 signal from 2003 onward indicates that the source is likely from established gas, coal, and coalbed methane mining and processing. This work demonstrates that space-based observations can identify anomalous CH4 emission source regions and quantify their emissions with the use of a transport model.

  3. Holocene dynamics of the Arctic's largest ice shelf.

    PubMed

    Antoniades, Dermot; Francus, Pierre; Pienitz, Reinhard; St-Onge, Guillaume; Vincent, Warwick F

    2011-11-22

    Ice shelves in the Arctic lost more than 90% of their total surface area during the 20th century and are continuing to disintegrate rapidly. The significance of these changes, however, is obscured by the poorly constrained ontogeny of Arctic ice shelves. Here we use the sedimentary record behind the largest remaining ice shelf in the Arctic, the Ward Hunt Ice Shelf (Ellesmere Island, Canada), to establish a long-term context in which to evaluate recent ice-shelf deterioration. Multiproxy analysis of sediment cores revealed pronounced biological and geochemical changes in Disraeli Fiord in response to the formation of the Ward Hunt Ice Shelf and its fluctuations through time. Our results show that the ice shelf was absent during the early Holocene and formed 4,000 years ago in response to climate cooling. Paleoecological data then indicate that the Ward Hunt Ice Shelf remained stable for almost three millennia before a major fracturing event that occurred ∼1,400 years ago. After reformation ∼800 years ago, freshwater was a constant feature of Disraeli Fiord until the catastrophic drainage of its epishelf lake in the early 21st century. PMID:22025693

  4. Largest meteor since Tunguska event explodes above Russian city

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2013-02-01

    The meteor that disintegrated in the atmosphere above Chelyabinsk, Russia, on 15 February, injuring more than 1000 people and causing widespread property damage from the shock wave, is the largest such incident since the 1908 Tunguska event in Siberia, according to Paul Chodas, research scientist in the Near-Earth Object (NEO) program office at NASA's Jet Propulsion Laboratory in Pasadena, Calif. During a 15 February teleconference, Chodas said it was an "incredible coincidence" that asteroid DA14 passed safely by Earth on the same day as the Chelyabinsk meteor; the 45-meter-wide asteroid sped by the planet at a distance of just 27,700 kilometers away on its closest approach. He stressed that the meteor was not related to DA14. The meteor "was coming from the wrong direction and at a completely different velocity," he said, noting that the orbit of the meteor went out to the asteroid belt while the orbit of DA14 "is very Earth-like, it does not go out so far."

  5. Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

    PubMed Central

    Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei

    2015-01-01

    ABSTRACT Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and

  6. New surprises in the largest magnetosphere of our solar system.

    PubMed

    Krupp, Norbert

    2007-10-12

    En route to its ultimate rendezvous with Pluto, the New Horizons spacecraft passed through the magnetic and plasma environment of Jupiter in February 2007. Onboard instruments collected high-resolution images, spectroscopic data, and information about charged particles. The results have revealed unusual structure and variation in Jupiter's plasma and large plasmoids that travel down the magnetotail. Data on Jupiter's aurora provide details of the interaction with the solar wind, and a major volcanic eruption from the moon Io was observed during the encounter. PMID:17932281

  7. Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins

    PubMed Central

    Batra, Jyoti; Tripathi, Shashank; Kumar, Amrita; Katz, Jacqueline M.; Cox, Nancy J.; Lal, Renu B.; Sambhara, Suryaprakash; Lal, Sunil K.

    2016-01-01

    A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins. PMID:26750153

  8. Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins.

    PubMed

    Batra, Jyoti; Tripathi, Shashank; Kumar, Amrita; Katz, Jacqueline M; Cox, Nancy J; Lal, Renu B; Sambhara, Suryaprakash; Lal, Sunil K

    2016-01-01

    A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins. PMID:26750153

  9. Cassini RADAR Observes Titan's Kraken Mare, The Largest Extraterrestrial Sea

    NASA Astrophysics Data System (ADS)

    Kirk, R. L.

    2013-12-01

    Synthetic aperture radar (SAR) imaging by Cassini's RADAR instrument beginning in 2006 revealed the presence of hundreds of hydrocarbon lakes and three large seas in Titan's north polar region [1,2]. Kraken Mare appeared to be the largest sea in IR images [3] but only the northern end was imaged by RADAR prior to 2012. High altitude SAR (HiSAR) observations on recent Titan flybys T84, T91, and T92 now provide complete coverage of Kraken and environs to ~50°N, at resolutions of 2-10 km. Backscatter statistics of the sea are 0.002 × 0.04, i.e., any return is substantially less than the noise and much less than typical dry land cross sections of 0.1-1.5. We conclude that Kraken is liquid filled to substantial depth over its entire extent. A surprising result is that the major seas all lie within a 1800 x 900 km 'box' (Fig.1). A third of the area inside this box (0.6 M km2) is sea, whereas the largest lake outside the box is only 0.005 M km2. Kraken's overall shape is complex (lobateness 4.4 vs 2.1 for Ligeia) but its 0.44 M km2 is divided by 20 to 160 km wide straits into 5 sub-basins that are elongated E-W across the box. Numerous straight shoreline segments parallel the edges of the box. Others parallel the 10°, 80°, and 170°W meridians, as do the rectangular drainage patterns mapped by [4]. These alignments suggest a key role for tectonics in creating the depressions occupied by the northern seas. Fensal and Aztlan (the 'lazy H' of dune-filled lows straddling the equator at longitudes 0°-70°W) form a similar pattern of elongated depressions connected by a narrow strait within a rectangular box. In contrast, Titan's small lakes are widely distributed. Multiple depressions with radar-dark floors have been identified near the south pole and hypothesized as former seabeds [5], but though similar in size to the northern seas, they are not aligned with one another or tightly clustered. Thus, the geography of the north and south poles will remain distinct even if

  10. Global assessment of nutrient loads to the world's largest lakes

    NASA Astrophysics Data System (ADS)

    Fink, Gabriel; Reder, Klara; Malsy, Marcus; Eisner, Stephanie; Flörke, Martina

    2015-04-01

    Lakes are essential resources of drinking water for a large part of mankind. Even so, most of the industrial and domestic waste water is discharged - often untreated - into rivers and streams that are finally the tributaries of these important freshwater bodies. Additionally, diffuse nutrient sources such as fertilizer and atmospheric deposition exacerbate existing algal blooms and low oxygen concentrations in rivers, lakes, and reservoirs. In this tense atmosphere of competing water uses, it is necessary to analyze all sources of pollution as well as their total contributions in order to protect these water bodies against deterioration. Finally, this is a general and urgently needed basis for developing recommendations for involved stakeholders and decision makers. Therefore, the project eartH2Observe, initiated and financed by the European Commission, creates the necessary and underlying quantitative and qualitative hydrological and water use data. In this context, information for global as well as for regional water resource assessments is being prepared based on new earth observations and an ensemble of global hydrological models. As a member of this ensemble, WaterGAP3 provides global estimates of lake water quality relevant parameters on a 5 arc minutes grid, namely total phosphorus and total nitrogen. These nutrient loads to lakes from different sources such as industrial fertilizer, organic fertilizer, domestic loads, atmospheric deposition, and urban surface runoff are estimated for the period 1990 to 2010 in a monthly time step. Whereas nutrient loads and their changes into numerous lakes worldwide are calculated, a special focus is set on nutrient loads into the large and shallow Lake Peipus, which is located between Estonia and Russia and subject to blooms of harmful cyanobacteria. We present estimates, trends, as well as sources of present nutrient loads (TN and TP) to the world's largest lakes with detailed insights to the Lake Peipus situation

  11. Functional characterization of the conserved amino acids in Pop1p, the largest common protein subunit of yeast RNases P and MRP

    PubMed Central

    Xiao, Shaohua; Hsieh, John; Nugent, Rebecca L.; Coughlin, Daniel J.; Fierke, Carol A.; Engelke, David R.

    2006-01-01

    RNase P and RNase MRP are ribonucleoprotein enzymes required for 5′-end maturation of precursor tRNAs (pre-tRNAs) and processing of precursor ribosomal RNAs, respectively. In yeast, RNase P and MRP holoenzymes have eight protein subunits in common, with Pop1p being the largest at >100 kDa. Little is known about the functions of Pop1p, beyond the fact that it binds specifically to the RNase P RNA subunit, RPR1 RNA. In this study, we refined the previous Pop1 phylogenetic sequence alignment and found four conserved regions. Highly conserved amino acids in yeast Pop1p were mutagenized by randomization and conditionally defective mutations were obtained. Effects of the Pop1p mutations on pre-tRNA processing, pre-rRNA processing, and stability of the RNA subunits of RNase P and MRP were examined. In most cases, functional defects in RNase P and RNase MRP in vivo were consistent with assembly defects of the holoenzymes, although moderate kinetic defects in RNase P were also observed. Most mutations affected both pre-tRNA and pre-rRNA processing, but a few mutations preferentially interfered with only RNase P or only RNase MRP. In addition, one temperature-sensitive mutation had no effect on either tRNA or rRNA processing, consistent with an additional role for RNase P, RNase MRP, or Pop1p in some other form. This study shows that the Pop1p subunit plays multiple roles in the assembly and function of of RNases P and MRP, and that the functions can be differentiated through the mutations in conserved residues. PMID:16618965

  12. The dust halo of Saturn's largest icy moon, Rhea.

    PubMed

    Jones, G H; Roussos, E; Krupp, N; Beckmann, U; Coates, A J; Crary, F; Dandouras, I; Dikarev, V; Dougherty, M K; Garnier, P; Hansen, C J; Hendrix, A R; Hospodarsky, G B; Johnson, R E; Kempf, S; Khurana, K K; Krimigis, S M; Krüger, H; Kurth, W S; Lagg, A; McAndrews, H J; Mitchell, D G; Paranicas, C; Postberg, F; Russell, C T; Saur, J; Seiss, M; Spahn, F; Srama, R; Strobel, D F; Tokar, R; Wahlund, J-E; Wilson, R J; Woch, J; Young, D

    2008-03-01

    Saturn's moon Rhea had been considered massive enough to retain a thin, externally generated atmosphere capable of locally affecting Saturn's magnetosphere. The Cassini spacecraft's in situ observations reveal that energetic electrons are depleted in the moon's vicinity. The absence of a substantial exosphere implies that Rhea's magnetospheric interaction region, rather than being exclusively induced by sputtered gas and its products, likely contains solid material that can absorb magnetospheric particles. Combined observations from several instruments suggest that this material is in the form of grains and boulders up to several decimetres in size and orbits Rhea as an equatorial debris disk. Within this disk may reside denser, discrete rings or arcs of material. PMID:18323452

  13. RNA gymnastics in mammalian signal recognition particle assembly.

    PubMed

    Wild, Klemens; Sinning, Irmgard

    2014-01-01

    More than one third of the cellular proteome is destined for incorporation into cell membranes or export from the cell. In all domains of life, the signal recognition particle (SRP) delivers these proteins to the membrane and protein traffic falls apart without SRP logistics. With the aid of a topogenic transport signal, SRP retrieves its cargo right at the ribosome, from where they are sorted to the translocation channel. Mammalian SRP is a ribonucleoprotein complex consisting of an SRP RNA of 300 nucleotides and 6 proteins bound to it. Assembly occurs in a hierarchical manner mainly in the nucleolus and only SRP54, which recognizes the signal sequence and regulates the targeting process, is added as the last component in the cytosol. Here we present an update on recent insights in the structure, function and dynamics of SRP RNA in SRP assembly with focus on the S domain, and present SRP as an example for the complex biogenesis of a rather small ribonucleoprotein particle. PMID:25692231

  14. RNA gymnastics in mammalian signal recognition particle assembly

    PubMed Central

    Wild, Klemens; Sinning, Irmgard

    2014-01-01

    Abstract More than one third of the cellular proteome is destined for incorporation into cell membranes or export from the cell. In all domains of life, the signal recognition particle (SRP) delivers these proteins to the membrane and protein traffic falls apart without SRP logistics. With the aid of a topogenic transport signal, SRP retrieves its cargo right at the ribosome, from where they are sorted to the translocation channel. Mammalian SRP is a ribonucleoprotein complex consisting of an SRP RNA of 300 nucleotides and 6 proteins bound to it. Assembly occurs in a hierarchical manner mainly in the nucleolus and only SRP54, which recognizes the signal sequence and regulates the targeting process, is added as the last component in the cytosol. Here we present an update on recent insights in the structure, function and dynamics of SRP RNA in SRP assembly with focus on the S domain, and present SRP as an example for the complex biogenesis of a rather small ribonucleoprotein particle. PMID:25692231

  15. Largest geomagnetic sudden commencement (SC) and interplanetary shock

    NASA Astrophysics Data System (ADS)

    Araki, Tohru

    2015-04-01

    The long term variation of amplitude of geomagnetic sudden commencements (SCs) is examined by checking old magnetograms at Kakioka (27.5 deg. geomagnetic latitude) and Alibag (10.3 deg.) and SC lists prepared by both stations. We found that the SC occurred on March 24, 1940 was largest since 1868. The amplitude is 310 nT at Alibag and larger than 273 nT at Kakioka. The magnetogram of Cape Town (-33.3 deg) was also available for this event which shows 164 nT amplitude. This SC occurred during the main phase of a large magnetic storm which has been interested as one of space weather events. The statistical analysis shows that the occurrence probability is less than 5 % for SCs with amplitude larger than 50 nT and less than 1 % for SCs larger than 100 nT at both Kakioka and Alibag. Large amplitude SCs tend to occur in the declining phase of the sun spot cycle as is reported for magnetic storms. Siscoe et al. (1968) firstly proposed the relationship for the solar wind dynamic pressure P and SC amplitude, dH as dH = C*d(P^0.5) where d(P^0.5) shows a jump of the square root of P associated with interplanetary shocks. If we take the proportionality constant C as 15 nT/(nPa)^0.5 and the 300 nT SC amplitude (dH) needs pressure jump from 2 nPa (assumed dynamic pressure in front of the shock) to 460 nPa. If the non-linear effect for magnetospheric compression is taken into account, a larger dynamic pressure will be needed for this large amplitude SC. On the other hand, the proportionality constant, C, might become larger for larger amplitude SC because C includes effects of electric currents induced in the earth. Larger amplitude SCs have larger time variation rate by which C becomes larger and the required dynamic pressure increase becomes smaller. We do not know which of the two competing processes is dominant but we consider that the linear estimation of the required dynamic pressure described above may be valid as the first order approximation.

  16. GIS learning tool for world's largest earthquakes and their causes

    NASA Astrophysics Data System (ADS)

    Chatterjee, Moumita

    The objective of this thesis is to increase awareness about earthquakes among people, especially young students by showing the five largest and two most predictable earthquake locations in the world and their plate tectonic settings. This is a geographic based interactive tool which could be used for learning about the cause of great earthquakes in the past and the safest places on the earth in order to avoid direct effect of earthquakes. This approach provides an effective way of learning for the students as it is very user friendly and more aligned to the interests of the younger generation. In this tool the user can click on the various points located on the world map which will open a picture and link to the webpage for that point, showing detailed information of the earthquake history of that place including magnitude of quake, year of past quakes and the plate tectonic settings that made this place earthquake prone. Apart from knowing the earthquake related information students will also be able to customize the tool to suit their needs or interests. Students will be able to add/remove layers, measure distance between any two points on the map, select any place on the map and know more information for that place, create a layer from this set to do a detail analysis, run a query, change display settings, etc. At the end of this tool the user has to go through the earthquake safely guidelines in order to be safe during an earthquake. This tool uses Java as programming language and uses Map Objects Java Edition (MOJO) provided by ESRI. This tool is developed for educational purpose and hence its interface has been kept simple and easy to use so that students can gain maximum knowledge through it instead of having a hard time to install it. There are lots of details to explore which can help more about what a GIS based tool is capable of. Only thing needed to run this tool is latest JAVA edition installed in their machine. This approach makes study more fun and

  17. Watching the Creation of Southern California's Largest Reservoir

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The new Diamond Valley Lake Reservoir near the city of Hemet in Riverside County is billed as the largest earthworks construction project in U.S.history. Construction began in 1995 and involved 31 million cubic meters of foundation excavation and 84 million cubic meters of embankment construction. This set of MISR images captures the most recent phase in the reservoir's activation. At the upper left is a natural-color view acquired by the instrument's vertical-viewing (nadir) camera on March 14, 2000 (Terra orbit 1273), shortly after the Metropolitan Water District began filling the reservoir with water from the Colorado River and Northern California. Water appears darker than the surrounding land. The image at the upper right was acquired nearly one year later on March 1, 2001 (Terra orbit 6399), and shows a clear increase in the reservoir's water content. When full, the lake will hold nearly a trillion liters of water.

    According to the Metropolitan Water District, the 7 kilometer x 3 kilometer reservoir nearly doubles Southern California's above-groundwater storage capacity. In addition to routine water management, Diamond Valley Lake is designed to provide protection against drought and a six-month emergency supply in the event of earthquake damage to a major aqueduct. In the face of electrical power shortages, it is also expected to reduce dependence on the pumping of water from northern mountains during the high-demand summer months. An unexpected result of site excavation was the uncovering of mastodon and mammoth skeletons along with bones from extinct species not previously thought to have been indigenous to the area, such as the giant long-horned bison and North American lion. A museum and interpretive center is being built to protect these finds.

    The lower MISR image, from May 20, 2001 (Terra orbit 7564), is a false-color view combining data from the instrument's 26-degree forward view (displayed as blue) with data from the 26-degree backward

  18. Final Status Survey for the Largest Decommissioning Project on Earth

    SciTech Connect

    Dubiel, R.W.; Miller, J.; Quayle, D.

    2006-07-01

    To assist the United States Department of Energy's (US DOE's) re-industrialization efforts at its gaseous diffusion site in Oak Ridge, Tennessee, known as the East Tennessee Technology Park (ETTP), the US DOE awarded a 6-year Decontamination and Decommissioning (D and D) contract to BNG America (formerly BNFL Inc.) in 1997. The ETTP 3-Building D and D Project included the removal and disposition of the materials and equipment from the K-33, K-31, and K-29 Gaseous Diffusion Plant buildings. The three buildings comprise more than 4.8 million square feet (446,000 square meters) of floor surface area and more than 350 million pounds (148 million kilograms) of hazardous and radioactively contaminated material, making it the largest nuclear D and D project in progress anywhere in the world. The logistical hurdles involved in a project of this scope and magnitude required an extensive amount of Engineering and Health Physics professionals. In order to accomplish the Final Status Survey (FSS) for a project of this scope, the speed and efficiency of automated survey equipment was essential. Surveys of floors, structural steel and ceilings up to 60 feet (18 meters) were required. The FSS had to be expanded to include additional remediation and surveys due to characterization surveys and assumptions regarding the nature and extent of contamination provided by the US DOE. Survey design and technical bases had to consider highly variable constituents; including uranium from depleted to low enrichment, variable levels of Technetium-99 and transuranic nuclides, which were introduced into the cascade during the 1960's when recycled uranium (RU) from Savannah River was re-enriched at the facility. The RU was transported to unexpected locations from leaks in the cascade by complex building ventilation patterns. The primary survey tool used for the post remediation and FSS was the Surface Contamination Monitor (SCM) and the associated Survey Information Management System (SIMS

  19. Drilling the Bushveld Complex- the world's largest layered mafic intrusion

    NASA Astrophysics Data System (ADS)

    Ashwal, L. D.; Webb, S. J.; Trumbull, R. B.

    2013-12-01

    The fact that surprising new discoveries can be made in layered mafic intrusions (e.g., subtle 100-150 m cyclicity in apparently homogeneous cumulates over 1000s of m) means that we are still in the first-order characterization phase of understanding these objects. Accordingly, we have secured funding from ICDP for a planning workshop to be held in Johannesburg in early 2014, aimed at scientific drilling of the Bushveld Complex, the world's largest layered mafic intrusion. Science objectives include, but are not limited to: 1. Magma chamber processes & melt evolution. How many melts/magmas/mushes were involved, what were their compositions and how did they interact? What, if anything, is missing from the Complex, and where did it go? Did Bushveld magmatism have an effect upon Earth's atmosphere at 2 Ga? 2. Crust-mantle interactions & origin of Bushveld granitoids. Are Bushveld granites & rhyolites crustal melts, differentiates from the mafic magmas or products of immiscibility? How can the evolved isotopic signatures in the mafic rocks (e.g., epsilon Nd to -8) be understood? 3. Origin of ore deposits. What were the relative roles of gravity settling, magma mixing, immiscibility and hydrothermal fluid transport in producing the PGE, Cr and V deposits? We have identified 3 potential drilling targets representing a total of ~12 km of drill core. Exact locations of drill sites are to be discussed at the workshop. Target A- East-Central Bushveld Complex. We propose 3 overlapping 3 km boreholes that will provide the first roof-to-floor continuous coverage of the Rustenburg Layered Suite. These boreholes will represent a curated, internationally available reference collection of Bushveld material for present and future research. Target B- Southeastern Bushveld Complex. We propose a single borehole of ~2 km depth, collared in Rooiberg felsite, and positioned to intersect the Roof Zone, Upper Zone, Main Zone and floor of the Complex. Amongst other things, this site will

  20. Smart-Geology for the World's largest fossil oyster reef

    NASA Astrophysics Data System (ADS)

    Dorninger, Peter; Nothegger, Clemens; Djuricic, Ana; Rasztovits, Sascha; Harzhauser, Mathias

    2014-05-01

    The geo-edutainment park "Fossilienwelt Weinviertel" at Stetten in Lower Austria exposes the world's largest fossil oyster biostrome. In the past decade, significant progress has been made in 3D digitizing sensor technology. To cope with the high amount of data, processing methods have been automated to a high degree. Consequently, we formulated the hypothesis that appropriate application of state-of-the-art 3D digitizing, data processing, and visualization technologies allows for a significant automation in paleontological prospection, making an evaluation of huge areas commercially feasible in both time and costs. We call the necessary processing steps "Smart Geology", being characterized by automation and large volumes of data. The Smart Geology project (FWF P 25883-N29) investigates three topics, 3D digitizing, automated geological and paleontological analysis and interpretation and finally investigating the applicability of smart devices for on-site accessibility of project data in order to support the two scientific hypotheses concerning the emerging process of the shell bed, i.e. was it formed by a tsunami or a major storm, and does it preserve pre- and post-event features. This contribution concentrates on the innovative and sophisticated 3D documentation and visualization processes being applied to virtualise approximately 15.000 fossil oysters at the approximately 25 by 17 m accessible shell bad. We decided to use a Terrestrial Laserscanner (TLS) for the determination of the geometrical 3D structures. The TLS achieves about 2 mm single point measurement accuracy. The scanning campaign provides a "raw" point cloud of approximately 1 bio. points at the respective area. Due to the scanning configuration used, the occurrence of occluded ares is minimized hence the full 3D structure of this unique site can be modelled. In addition, approximately 300 photos were taken with a nominal resolution of 0.6 mm per pixel. Sophisticated artificial lightning (close to

  1. The Largest Holocene Eruption of the Central Andes Found

    NASA Astrophysics Data System (ADS)

    Fernandez-Turiel, J.; Rodriguez-Gonzalez, A.; Saavedra, J.; Perez-Torrado, F.; Carracedo, J.; Osterrieth, M.; Carrizo, J.; Esteban, G.

    2013-12-01

    We present new data and interpretation about a major eruption -spreading ˜110 km3 ashes over 440.000 km2- long thought to have occurred around 4200 years ago in the Cerro Blanco Volcanic Complex (CBVC) in NW Argentina. This eruption may be the biggest during the past five millennia in the Central Volcanic Zone of the Andes, and possibly one of the largest Holocene eruptions in the world. The environmental effects of this voluminous eruption are still noticeable, as evidenced by the high content of arsenic and other trace elements in the groundwaters of the Chacopampean Plain. The recognition of this significant volcanic event may shed new light on interpretations of critical changes observed in the mid-Holocene paleontological and archaeological records, and offers researchers an excellent, extensive regional chronostratigraphic marker for reconstructing mid-Holocene geological history over a wide geographical area of South America. More than 100 ashes were sampled in Argentina, Chile and Uruguay during different field campaigns. Ash samples were characterized by scanning electron microscope (SEM), X-ray diffraction (XRD), grain size distributions laser diffraction, and geochemically by electron microprobe (EMPA) and laser ablation-HR-ICP-MS. New and published 14C ages were calibrated to calendar years BP. The age of the most recent CBVC eruption is 4407-4093 cal y BP, indirectly dated by 14C of associated organic sediment within the lower part of a proximal fall deposit of this event (26°53'16.05"S-67°44'48.68"W). This is the youngest record of a major volcanic event in the Southern Puna. This age is consistent with other radiocarbon dates of organic matter in palaeosols underlying or overlying distal ash fall deposits. Based on their products, all of rhyolitic composition, we have distinguished 8 main episodes during the evolution of the most recent CBVC eruption: 1) the eruption began with a white rhyolite lava dome extrusion; 2) followed by a Plinian

  2. ALMA Partners Break Ground on World's Largest Millimeter Wavelength Telescope

    NASA Astrophysics Data System (ADS)

    2003-11-01

    Scientists and dignitaries from North America, Europe, and Chile broke ground today (Thursday, November 6, 2003) on what will be the world's largest, most sensitive radio telescope operating at millimeter wavelengths. ALMA - the Atacama Large Millimeter Array - will be a single instrument composed of 64 high-precision antennas located on the Chajnantor plain of the Chilean Andes in the District of San Pedro de Atacama, 16,500 feet (5,000 meters) above sea level. ALMA's primary function will be to observe and image with unprecedented clarity the enigmatic cold regions of the Universe, which are optically dark, yet shine brightly in the millimeter portion of the electromagnetic spectrum. ALMA Array Artist's Conception of ALMA Array in Compact Configuration (Click on Image for Larger Version) Other Images Available: Artist's conception of the antennas for the Atacama Large Millimeter Array Moonrise over ALMA test equipment near Cerro Chajnantor, Chile VertexRSI antenna at the VLA test site The Atacama Large Millimeter Array is an international astronomy facility. ALMA is an equal partnership between Europe and North America, in cooperation with the Republic of Chile, and is funded in North America by the U.S. National Science Foundation (NSF) in cooperation with the National Research Council of Canada (NRC), and in Europe by the European Southern Observatory (ESO) and Spain. ALMA construction and operations are led on behalf of North America by the National Radio Astronomy Observatory (NRAO), which is managed by Associated Universities, Inc. (AUI), and on behalf of Europe by ESO. "The U.S. National Science Foundation joins today with our North American partner, Canada, and with the European Southern Observatory, Spain, and Chile to prepare for a spectacular new instrument," said Dr. Rita Colwell, director of the U.S. National Science Foundation. "The Atacama Large Millimeter Array will expand our vision of the Universe with "eyes" that pierce the shrouded mantles of

  3. Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses.

    PubMed

    Ngai, Karry L K; Chan, Martin C W; Chan, Paul K S

    2013-01-01

    Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production. PMID:23750226

  4. Isolation and characterization of a Xenopus laevis C protein cDNA: structure and expression of a heterogeneous nuclear ribonucleoprotein core protein.

    PubMed Central

    Preugschat, F; Wold, B

    1988-01-01

    The C proteins are major components of heterogeneous nuclear ribonucleoprotein complexes in nuclei of vertebrate cells. To begin to describe their structure, expression, and function we isolated and determined the DNA sequence of Xenopus laevis C protein cDNA clones. The protein predicted from the DNA sequence has a molecular mass of 30,916 kDa and is very similar to its human counterpart. Although mammalian genomes contain many copies of C protein sequence, the Xenopus genome contains few copies. When C protein RNA was synthesized in vitro and microinjected into stage-VI Xenopus oocytes, newly synthesized C proteins were efficiently localized in the nucleus. In vitro rabbit reticulocyte lysate and in vivo Xenopus oocyte translation systems both produce from a single mRNA two discrete polypeptide species that accumulate in a ratio similar to that of mammalian C1 and C2 proteins in vivo. Images PMID:2904678

  5. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition

    PubMed Central

    Kondo, Yasushi; Oubridge, Chris; van Roon, Anne-Marie M; Nagai, Kiyoshi

    2015-01-01

    U1 snRNP binds to the 5′ exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein–protein and RNA-protein interactions within U1 snRNP, and show how the 5′ splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5′-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5′-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5′-splice sites. DOI: http://dx.doi.org/10.7554/eLife.04986.001 PMID:25555158

  6. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.

    PubMed

    Kondo, Yasushi; Oubridge, Chris; van Roon, Anne-Marie M; Nagai, Kiyoshi

    2015-01-01

    U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites. PMID:25555158

  7. Splicing remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex components.

    PubMed

    Zhang, Zuo; Krainer, Adrian R

    2007-07-10

    Pre-mRNA splicing not only removes introns and joins exons to generate spliced mRNA but also results in remodeling of the spliced messenger ribonucleoprotein, influencing various downstream events. This remodeling includes the loading of an exon-exon junction complex (EJC). It is unclear how the spliceosome recruits the EJC onto the mRNA and whether EJC formation or EJC components are required for pre-mRNA splicing. Here we immunodepleted the EJC core component eIF4A3 from HeLa cell nuclear extract and found that eIF4A3 is dispensable for pre-mRNA splicing in vitro. However, eIF4A3 is required for the splicing-dependent loading of the Y14/Magoh heterodimer onto mRNA, and this activity of human eIF4A3 is also present in the Drosophila ortholog. Surprisingly, the loading of six other EJC components was not affected by eIF4A3 depletion, suggesting that their binding to mRNA involves different or redundant pathways. Finally, we found that the assembly of the EJC onto mRNA occurs at the late stages of the splicing reaction and requires the second-step splicing and mRNA-release factor HRH1/hPrp22. The EJC-dependent and -independent recruitment of RNA-binding proteins onto mRNA suggests a role for the EJC in messenger ribonucleoprotein remodeling involving interactions with other proteins already bound to the pre-mRNA, which has implications for nonsense-mediated mRNA decay and other mRNA transactions. PMID:17606899

  8. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution

    PubMed Central

    König, Julian; Zarnack, Kathi; Rot, Gregor; Curk, Tomaž; Kayikci, Melis; Zupan, Blaž; Turner, Daniel J.; Luscombe, Nicholas M.; Ule, Jernej

    2010-01-01

    In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C. Despite their abundance however, it remained unclear whether these particles control pre-mRNA processing. Here, we developed individual-nucleotide resolution UV-cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. iCLIP data demonstrate that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. hnRNP particles assemble on both introns and exons, but remain generally excluded from splice sites. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an ‘RNA map’ indicates how the positioning of hnRNP particles determines their effect on inclusion of alternative exons. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes. PMID:20601959

  9. Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex.

    PubMed

    Vijayapalani, Paramasivan; Chen, Jeff Chien-Fu; Liou, Ming-Ru; Chen, Hsin-Chuan; Hsu, Yau-Heiu; Lin, Na-Sheng

    2012-01-01

    Bamboo mosaic virus (BaMV) satellite RNA (satBaMV) depends on BaMV for its replication and encapsidation. SatBaMV-encoded P20 protein is an RNA-binding protein that facilitates satBaMV systemic movement in co-infected plants. Here, we examined phosphorylation of P20 and its regulatory functions. Recombinant P20 (rP20) was phosphorylated by host cellular kinase(s) in vitro, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mutational analyses revealed Ser-11 as the phosphorylation site. The phosphor-mimic rP20 protein interactions with satBaMV-translated mutant P20 were affected. In overlay assay, the Asp mutation at S11 (S11D) completely abolished the self-interaction of rP20 and significantly inhibited the interaction with both the WT and S11A rP20. In chemical cross-linking assays, S11D failed to oligomerize. Electrophoretic mobility shift assay and subsequent Hill transformation analysis revealed a low affinity of the phospho-mimicking rP20 for satBaMV RNA. Substantial modulation of satBaMV RNA conformation upon interaction with nonphospho-mimic rP20 in circular dichroism analysis indicated formation of stable satBaMV ribonucleoprotein complexes. The dissimilar satBaMV translation regulation of the nonphospho- and phospho-mimic rP20 suggests that phosphorylation of P20 in the ribonucleoprotein complex converts the translation-incompetent satBaMV RNA to messenger RNA. The phospho-deficient or phospho-mimicking P20 mutant of satBaMV delayed the systemic spread of satBaMV in co-infected Nicotiana benthamiana with BaMV. Thus, satBaMV likely regulates the formation of satBaMV RNP complex during co-infection in planta. PMID:21965537

  10. Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells.

    PubMed

    Chang, Elizabeth T; Parekh, Palak R; Yang, Qingyuan; Nguyen, Duc M; Carrier, France

    2016-03-01

    The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3'UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells. PMID:26824423

  11. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

    PubMed

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-04-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. PMID:26848818

  12. Borna Disease Virus Matrix Protein Is an Integral Component of the Viral Ribonucleoprotein Complex That Does Not Interfere with Polymerase Activity▿

    PubMed Central

    Chase, Geoffrey; Mayer, Daniel; Hildebrand, Antonia; Frank, Ronald; Hayashi, Yohei; Tomonaga, Keizo; Schwemmle, Martin

    2007-01-01

    We have recently shown that the matrix protein M of Borna disease virus (BDV) copurifies with the affinity-purified nucleoprotein (N) from BDV-infected cells, suggesting that M is an integral component of the viral ribonucleoprotein complex (RNP). However, further studies were hampered by the lack of appropriate tools. Here we generated an M-specific rabbit polyclonal antiserum to investigate the intracellular distribution of M as well as its colocalization with other viral proteins in BDV-infected cells. Immunofluorescence analysis revealed that M is located both in the cytoplasm and in nuclear punctate structures typical for BDV infection. Colocalization studies indicated an association of M with nucleocapsid proteins in these nuclear punctate structures. In situ hybridization analysis revealed that M also colocalizes with the viral genome, implying that M associates directly with viral RNPs. Biochemical studies demonstrated that M binds specifically to the phosphoprotein P but not to N. Binding of M to P involves the N terminus of P and is independent of the ability of P to oligomerize. Surprisingly, despite P-M complex formation, BDV polymerase activity was not inhibited but rather slightly elevated by M, as revealed in a minireplicon assay. Thus, unlike M proteins of other negative-strand RNA viruses, BDV-M seems to be an integral component of the RNPs without interfering with the viral polymerase activity. We propose that this unique feature of BDV-M is a prerequisite for the establishment of BDV persistence. PMID:17079312

  13. Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures

    PubMed Central

    Uribe, Diana J.; Guo, Kexiao; Shin, Yoon-Joo; Sun, Daekyu

    2011-01-01

    The human vascular endothelial growth factor (VEGF) promoter contains a polypurine/polypyrimidine (pPu/pPy) tract that is known to play a critical role in its transcriptional regulation. This pPu/pPy tract undergoes a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We studied the interaction of the cytosine-rich (C-rich) and guanine-rich (G-rich) strands of this tract with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of VEGF. Using chromatin immunoprecipitation (ChIP) assay for our in vivo studies and electrophoretic mobility shift assay (EMSA) for our in vitro studies, we demonstrated that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the VEGF promoter. The small interfering RNA (siRNA)-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within the pPu/pPy tract will provide new insight into mechanisms of transcriptional regulation of the VEGF gene. PMID:21466159

  14. Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution

    SciTech Connect

    Chang, Yuan-I; Hsu, Sheng-Chieh; Chau, Gar-Yang; Huang, Chi-Ying F.; Sung, Jung-Sung; Hua, Wei-Kai; Lin, Wey-Jinq

    2011-01-21

    Research highlights: {yields} Verifying by direct methylation assay the substrate sites of PRMT1 in the hnRNP K protein. {yields} Identifying the preferred PMRT1 methylation regions in hnRNP K by kinetic analysis. {yields} Linking methylation in regulating nuclear localization of hnRNP K. -- Abstract: Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates, here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.

  15. Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells.

    PubMed

    Avilov, Sergiy; Magnus, Julie; Cusack, Stephen; Naffakh, Nadia

    2016-01-01

    Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells. PMID:26978069

  16. Promoter-associated small double-stranded RNA interacts with heterogeneous nuclear ribonucleoprotein A2/B1 to induce transcriptional activation.

    PubMed

    Hu, Jia; Chen, Zhong; Xia, Ding; Wu, Jia; Xu, Hua; Ye, Zhang-Qun

    2012-11-01

    Several recent reports have demonstrated that small activating dsRNA [double-stranded RNA; saRNA (small activating dsRNA)] complementary to promoter regions can up-regulate gene expression in mammalian cells, a phenomenon termed RNAa (RNA activation). However, the mechanism of RNAa remains obscure with regard to what is the target molecule for promoter-targeted saRNA and what are the proteins involved in this process. p21Waf1/Cip1 (p21) [CDKN1A (cyclin-dependent kinase inhibitor 1A)], an important tumour suppressor gene, is among the genes that can be activated by RNAa in tumour cells. In the present study, we provide direct evidence that p21 promoter-targeted saRNA interact with its intended target on the p21 promoter to activate p21 expression. This process is associated with recruitment of RNA polymerase II and AGO2 (argonaute 2) protein to the saRNA-target site. Additionally, we found that several hnRNPs (heterogeneous nuclear ribonucleoproteins) (A1, A2/B1 and C1/C2) are associated with saRNA. Further studies show that hnRNPA2/B1 interacts with the saRNA in vivo and in vitro and is required for RNAa activity. These findings indicate that RNAa results from specific targeting of promoters and reveals additional mechanistic details of RNAa. PMID:23035981

  17. Heterogeneous ribonucleoprotein R regulates arylalkylamine N-acetyltransferase synthesis via internal ribosomal entry site-mediated translation in a circadian manner.

    PubMed

    Lee, Hwa-Rim; Kim, Tae-Don; Kim, Hyo-Jin; Jung, Youngseob; Lee, Dohyun; Lee, Kyung-Ha; Kim, Do-Yeon; Woo, Kyung-Chul; Kim, Kyong-Tai

    2015-11-01

    Rhythmic arylalkylamine N-acetyltransferase (AANAT) synthesis is a prominent circadian-controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA-binding protein hnRNP R widely interacted with the 5' untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine-tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap-independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R-bound AANAT mRNA. PMID:26444903

  18. Nucleolin and heterogeneous nuclear ribonucleoprotein C proteins specifically interact with the 3'-untranslated region of amyloid protein precursor mRNA.

    PubMed

    Zaidi, S H; Malter, J S

    1995-07-21

    The central nervous system deposition by neurons and glia of beta A4 amyloid protein is an important contributing factor to the development of Alzheimer's disease. Amyloidogenic cells overexpress amyloid precursor protein (APP) mRNAs suggesting a transcriptional or post-transcriptional defect may contribute to this process. We have previously shown that APP mRNAs display regulated stability which is dependent on a 29-base element within the 3'-untranslated region (UTR). This domain specifically interacted with several cytoplasmic RNA-binding proteins. We have purified these APP RNA-binding proteins from a human T-cell leukemia and demonstrate that five cytoplasmic proteins of 70, 48, 47, 39, and 38 kDa form the previously observed APP RNA protein complexes. Amino acid sequence analyses showed that the 70-, 48-, and 47-kDa proteins were fragments of nucleolin and that the 39- and 38-kDa proteins were heterogeneous nuclear ribonucleoprotein (hnRNP) C protein. Northwestern and Western blot analyses of purified material further confirmed these data. Nucleolin protein is known to shuttle between the nucleus and cytoplasm but hnRNP C has not been reported within the cytoplasm. This report of sequence specific, mRNA binding by nucleolin and hnRNP C suggests that these proteins participate in the post-transcriptional regulation of APP mRNA through 3'-UTR, site-specific interactions. PMID:7615529

  19. Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1.

    PubMed

    Jain, Niyati; Morgan, Christopher E; Rife, Brittany D; Salemi, Marco; Tolbert, Blanton S

    2016-01-29

    Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3' acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein. PMID:26607354

  20. Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

    PubMed Central

    Monette, Anne; Ajamian, Lara; López-Lastra, Marcelo; Mouland, Andrew J.

    2009-01-01

    Human immunodeficiency virus type 1 (HIV-1) co-opts host proteins and cellular machineries to its advantage at every step of the replication cycle. Here we show that HIV-1 enhances heterogeneous nuclear ribonucleoprotein (hnRNP) A1 expression and promotes the relocalization of hnRNP A1 to the cytoplasm. The latter was dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of the FG-repeat nuclear pore glycoprotein p62. hnRNP A1 and vRNA remain colocalized in the cytoplasm supporting a post-nuclear function during the late stages of HIV-1 replication. Consistently, we show that hnRNP A1 acts as an internal ribosomal entry site trans-acting factor up-regulating internal ribosome entry site-mediated translation initiation of the HIV-1 vRNA. The up-regulation and cytoplasmic retention of hnRNP A1 by HIV-1 would ensure abundant expression of viral structural proteins in cells infected with HIV-1. PMID:19737937

  1. Overexpression of an Arabidopsis heterogeneous nuclear ribonucleoprotein gene, AtRNP1, affects plant growth and reduces plant tolerance to drought and salt stresses.

    PubMed

    Wang, Zhenyu; Zhao, Xiuyang; Wang, Bing; Liu, Erlong; Chen, Ni; Zhang, Wei; Liu, Heng

    2016-04-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) participate in diverse regulations of plant growth and environmental stress responses. In this work, an Arabidopsis hnRNP of unknown function, AtRNP1, was investigated. We found that AtRNP1 gene is highly expressed in rosette and cauline leaves, and slightly induced under drought, salt, osmotic and ABA stresses. AtRNP1 protein is localized to both the nucleus and cytoplasm. We performed homologous overexpression of AtRNP1 and found that the transgenic plants showed shortened root length and plant height, and accelerated flowering. In addition, the transgenic plants also showed reduced tolerance to drought, salt, osmotic and ABA stresses. Further studies revealed that under both normal and stress conditions, the proline contents in the transgenic plants are markedly decreased, associated with reduced expression levels of a proline synthase gene and several stress-responsive genes. These results suggested that the overexpression of AtRNP1 negatively affects plant growth and abiotic stress tolerance. PMID:26923071

  2. U1 small nuclear ribonucleoproteins (snRNPs) aggregate in Alzheimer’s disease due to autosomal dominant genetic mutations and trisomy 21

    PubMed Central

    2014-01-01

    Background We recently identified U1 small nuclear ribonucleoprotein (snRNP) tangle-like aggregates and RNA splicing abnormalities in sporadic Alzheimer’s disease (AD). However little is known about snRNP biology in early onset AD due to autosomal dominant genetic mutations or trisomy 21 in Down syndrome. Therefore we investigated snRNP biochemical and pathologic features in these disorders. Findings We performed quantitative proteomics and immunohistochemistry in postmortem brain from genetic AD cases. Electron microscopy was used to characterize ultrastructural features of pathologic aggregates. U1-70k and other snRNPs were biochemically enriched in the insoluble fraction of human brain from subjects with presenilin 1 (PS1) mutations. Aggregates of U1 snRNP-immunoreactivity formed cytoplasmic tangle-like structures in cortex of AD subjects with PS1 and amyloid precursor protein (APP) mutations as well as trisomy 21. Ultrastructural analysis with electron microscopy in an APP mutation case demonstrated snRNP immunogold labeling of paired helical filaments (PHF). Conclusions These studies identify U1 snRNP pathologic changes in brain of early onset genetic forms of AD. Since dominant genetic mutations and trisomy 21 result in dysfunctional amyloid processing, the findings suggest that aberrant β-amyloid processing may influence U1 snRNP aggregate formation. PMID:24773620

  3. Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells

    PubMed Central

    Avilov, Sergiy; Magnus, Julie; Cusack, Stephen; Naffakh, Nadia

    2016-01-01

    Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells. PMID:26978069

  4. Heterogeneous nuclear ribonucleoprotein (hnRNP) F is a novel component of oligodendroglial RNA transport granules contributing to regulation of myelin basic protein (MBP) synthesis.

    PubMed

    White, Robin; Gonsior, Constantin; Bauer, Nina M; Krämer-Albers, Eva-Maria; Luhmann, Heiko J; Trotter, Jacqueline

    2012-01-13

    Myelin basic protein (MBP) is a major component of central nervous system (CNS) myelin. The absence of MBP results in the loss of almost all compact myelin in the CNS. MBP mRNA is sorted into RNA granules that are transported to the periphery of oligodendrocytes in a translationally inactive state. A central mediator of this transport process is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 that binds to the cis-acting A2-response element in the 3'UTR of MBP mRNA. Recently, we found that activation of the Src family nonreceptor tyrosine kinase Fyn in oligodendrocytes leads to phosphorylation of hnRNP A2 and to increased translation of MBP mRNA. Here, we identify the RNA-binding protein hnRNP F as a novel component of MBP mRNA transport granules. It is associated with hnRNP A2 and MBP mRNA in cytoplasmic granular structures and is involved in post-transcriptional regulation of MBP expression. Fyn kinase activity results in phosphorylation of hnRNP F in the cytoplasm and its release from MBP mRNA and RNA granules. Our results define hnRNP F as a regulatory element of MBP expression in oligodendrocytes and imply an important function of hnRNP F in the control of myelin synthesis. PMID:22128153

  5. Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells

    PubMed Central

    Chang, Elizabeth T.; Parekh, Palak R.; Yang, Qingyuan; Nguyen, Duc M.; Carrier, France

    2016-01-01

    The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3′UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells. PMID:26824423

  6. Centromere Protein (CENP)-W Interacts with Heterogeneous Nuclear Ribonucleoprotein (hnRNP) U and May Contribute to Kinetochore-Microtubule Attachment in Mitotic Cells

    PubMed Central

    Chun, Younghwa; Kim, Raehyung; Lee, Soojin

    2016-01-01

    Background Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. Results We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. Conclusion Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis. PMID:26881882

  7. Epidermal growth factor increases the interaction between nucleolin and heterogeneous nuclear ribonucleoprotein K/poly(C) binding protein 1 complex to regulate the gastrin mRNA turnover.

    PubMed

    Lee, Pin-Tse; Liao, Pao-Chi; Chang, Wen-Chang; Tseng, Joseph T

    2007-12-01

    Gastrin, a gastrointestinal hormone responsible for gastric acid secretion, has been confirmed as a growth factor for gastrointestinal tract malignancies. High expression of gastrin mRNA was observed in pancreatic and colorectal cancer; however, the mechanism is unclear. Epidermal growth factor (EGF) was found to increase gastrin mRNA stability, indicating mRNA turnover regulation mechanism is involved in the control of gastrin mRNA expression. Using biotin-labeled RNA probe pull-down assay combined with mass spectrometry analysis, we identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and poly(C) binding protein 1 (PCBP1) bound with the C-rich region in gastrin mRNA 3' untranslated region. Nucleolin bound with the AGCCCU motif and interacted with hnRNP K were also demonstrated. Under EGF treatment, we observed the amount of nucleolin interacting with hnRNP K and gastrin mRNA increased. Using small interfering RNA technology to define their functional roles, we found hnRNP K, PCBP1, and nucleolin were all responsible for stabilizing gastrin mRNA. Moreover, nucleolin plays a crucial role in mediating the increased gastrin mRNA stability induced by EGF signaling. Besides, we also observed hnRNP K/PCBP1 complex bound with the C-rich region in the gastrin mRNA increased nucleolin binding with gastrin mRNA. Finally, a novel binding model was proposed. PMID:17928403

  8. Chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as the molecular target of quercetin in its anti-cancer effects in PC-3 cells.

    PubMed

    Ko, Chia-Chen; Chen, Yun-Ju; Chen, Chih-Ta; Liu, Yu-Chih; Cheng, Fong-Chi; Hsu, Kai-Chao; Chow, Lu-Ping

    2014-08-01

    Quercetin, a flavonoid abundantly present in plants, is widely used as a phytotherapy in prostatitis and prostate cancer. Although quercetin has been reported to have a number of therapeutic effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and in vitro binding experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules after treatment of cells with quercetin for up to 48 h, and the levels of cIAP1 (cellular inhibitor of apoptosis), an internal ribosome entry site translation-dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1, insights that were obtained using a chemical proteomics strategy. PMID:24962584

  9. Heterogeneous Nuclear Ribonucleoprotein (hnRNP) F Is a Novel Component of Oligodendroglial RNA Transport Granules Contributing to Regulation of Myelin Basic Protein (MBP) Synthesis*

    PubMed Central

    White, Robin; Gonsior, Constantin; Bauer, Nina M.; Krämer-Albers, Eva-Maria; Luhmann, Heiko J.; Trotter, Jacqueline

    2012-01-01

    Myelin basic protein (MBP) is a major component of central nervous system (CNS) myelin. The absence of MBP results in the loss of almost all compact myelin in the CNS. MBP mRNA is sorted into RNA granules that are transported to the periphery of oligodendrocytes in a translationally inactive state. A central mediator of this transport process is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 that binds to the cis-acting A2-response element in the 3′UTR of MBP mRNA. Recently, we found that activation of the Src family nonreceptor tyrosine kinase Fyn in oligodendrocytes leads to phosphorylation of hnRNP A2 and to increased translation of MBP mRNA. Here, we identify the RNA-binding protein hnRNP F as a novel component of MBP mRNA transport granules. It is associated with hnRNP A2 and MBP mRNA in cytoplasmic granular structures and is involved in post-transcriptional regulation of MBP expression. Fyn kinase activity results in phosphorylation of hnRNP F in the cytoplasm and its release from MBP mRNA and RNA granules. Our results define hnRNP F as a regulatory element of MBP expression in oligodendrocytes and imply an important function of hnRNP F in the control of myelin synthesis. PMID:22128153

  10. Morphological details in bloodstain particles.

    PubMed

    De Wael, K; Lepot, L

    2015-01-01

    During the commission of crimes blood can be transferred to the clothing of the offender or on other crime related objects. Bloodstain particles are sub-millimetre sized flakes that are lost from dried bloodstains. The nature of these red particles is easily confirmed using spectroscopic methods. In casework, bloodstain particles showing highly detailed morphological features were observed. These provided a rationale for a series of experiments described in this work. It was found that the "largest" particles are shed from blood deposited on polyester and polyamide woven fabrics. No particles are lost from the stains made on absorbent fabrics and from those made on knitted fabrics. The morphological features observed in bloodstain particles can provide important information on the substrates from which they were lost. PMID:25437904