Heterogeneous nuclear ribonucleoprotein D/AUF1 interacts with ...

Indian Academy of Sciences (India)

SEARCHU

Ribonucleic acids (RNAs) in cells are bound to proteins. Heterogeneous nuclear ribonucleoprotein (hnRNP) is one of the representative proteins bound to RNAs in eukaryotic cells. More than 30 hnRNPs have been determined to exist in human nuclei, and are referred to as hnRNPs A1 through U (Choi and Dreyfuss 1984; ...

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

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Pitzonka, Laura; Ullas, Sumana; Chinnam, Meenalakshmi; Povinelli, Benjamin J; Fisher, Daniel T; Golding, Michelle; Appenheimer, Michelle M; Nemeth, Michael J; Evans, Sharon; Goodrich, David W

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

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Laura Pitzonka

Full Text Available Co-transcriptionally assembled ribonucleoprotein (RNP complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

Comparison of the ribonucleoproteins of different rabies virus serotypes by radioimmunoassay

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Bruns, M; Dietzschold, B; Schneider, L G; Cox, J H [Federal Research Inst. for Animal Virus Diseases, Tuebingen (Germany, F.R.)

1977-12-01

Radioimmunoassay (RIA) provides a sensitive serological procedure for detecting rabies virus ribonucleoprotein (RNP) as well as its specific antibodies. RIA was carried out using highly purified RNPs labelled by the chloramine-T method. This paper describes optimal conditions for iodination of RNP with high specific activity. The optimal concentrations of /sup 125/I, RNP, chloramine-T, and reducing agent as well as the effect of pH on the reaction were investigated. RIA proved to be extremely sensitive for detection of homologous antibodies. In competition experiments the part-relationship of the group-specific RNPs of the three rabies virus serotypes (HEP, MOK, and LBV) was confirmed.

Spinal Muscular Atrophy: From Defective Chaperoning of snRNP Assembly to Neuromuscular Dysfunction

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Maia Lanfranco

2017-06-01

Full Text Available Spinal Muscular Atrophy (SMA is a neuromuscular disorder that results from decreased levels of the survival motor neuron (SMN protein. SMN is part of a multiprotein complex that also includes Gemins 2–8 and Unrip. The SMN-Gemins complex cooperates with the protein arginine methyltransferase 5 (PRMT5 complex, whose constituents include WD45, PRMT5 and pICln. Both complexes function as molecular chaperones, interacting with and assisting in the assembly of an Sm protein core onto small nuclear RNAs (snRNAs to generate small nuclear ribonucleoproteins (snRNPs, which are the operating components of the spliceosome. Molecular and structural studies have refined our knowledge of the key events taking place within the crowded environment of cells and the numerous precautions undertaken to ensure the faithful assembly of snRNPs. Nonetheless, it remains unclear whether a loss of chaperoning in snRNP assembly, considered as a “housekeeping” activity, is responsible for the selective neuromuscular phenotype in SMA. This review thus shines light on in vivo studies that point toward disturbances in snRNP assembly and the consequential transcriptome abnormalities as the primary drivers of the progressive neuromuscular degeneration underpinning the disease. Disruption of U1 snRNP or snRNP assembly factors other than SMN induces phenotypes that mirror aspects of SMN deficiency, and splicing defects, described in numerous SMA models, can lead to a DNA damage and stress response that compromises the survival of the motor system. Restoring the correct chaperoning of snRNP assembly is therefore predicted to enhance the benefit of SMA therapeutic modalities based on augmenting SMN expression.

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

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Reimer, G.; Raska, I.; Scheer, U.; Tan, E.M.

1988-01-01

Certain autoimmune sera contain antibodies against a nucleolar ribonucleotprotein particle associated with 7-2-RNA. In this study, the authors showed by immunofluorescence microscopy that antibodies reactive with 7-2-ribonucleoprotein immunolocalized in the granular regions of actinomycin D and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)--segregated nucleoli from Vero cells. By electron microscopic immunocytochemistry, antigen-antibody complexes were located in the granular component of transcriptionally active nucleoli from rat liver hepatocytes and HeLa cells. Anti-7-2-RNP antibodies from two autoimmune sera immunoprecipitated a major protein of M r 40,000 from [ 35 S] methionine-labeled HeLa cell extract. The immunolocalization data suggest that 7-2-ribonucleoprotein may be involved in stages of ribosome biogenesis which take place in the granular component of the nucleolus, i.e., assembly, maturation, and/or transport of preribosomes

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

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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.

The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion.

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Singh, Guramrit; Pratt, Gabriel; Yeo, Gene W; Moore, Melissa J

2015-01-01

Throughout their lifetimes, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Since the discovery of the first mRNP component more than 40 years ago, what is known as the mRNA interactome now comprises >1,000 proteins. These proteins bind mRNAs in myriad ways with varying affinities and stoichiometries, with many assembling onto nascent RNAs in a highly ordered process during transcription and precursor mRNA (pre-mRNA) processing. The nonrandom distribution of major mRNP proteins observed in transcriptome-wide studies leads us to propose that mRNPs are organized into three major domains loosely corresponding to 5' untranslated regions (UTRs), open reading frames, and 3' UTRs. Moving from the nucleus to the cytoplasm, mRNPs undergo extensive remodeling as they are first acted upon by the nuclear pore complex and then by the ribosome. When not being actively translated, cytoplasmic mRNPs can assemble into large multi-mRNP assemblies or be permanently disassembled and degraded. In this review, we aim to give the reader a thorough understanding of past and current eukaryotic mRNP research.

Isolation and characterization of the heterogeneous nuclear RNA-ribonucleoprotein complex

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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 [ 35 S]methionine-labeled proteins which become crosslinked to poly(A) + 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 ∼10 kb. The major steady state labeled [ 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

Perturbation of Ribosome Biogenesis Drives Cells into Senescence through 5S RNP-Mediated p53 Activation

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Kazuho Nishimura

2015-03-01

Full Text Available The 5S ribonucleoprotein particle (RNP complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei.

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Kamina, Anyango D; Jaremko, Daniel; Christen, Linda; Williams, Noreen

2017-01-01

Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei , the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T . brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei . IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of

MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1

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Ziaei, Samira; Shimada, Naoko; Kucharavy, Herman; Hubbard, Karen

2012-01-01

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence. -- Highlights: ► MNK1 and not MAPKAPK2 phosphorylates hnRNP A1. ► MNK1 has elevated levels in senescent cells, this has not been reported previously. ► MNK1 activity induces cytoplasmic accumulation of hnRNP A1 in senescent cells. ► Altered cytoplasmic localization of hnRNP A1 may alter gene expression patterns. ► Our studies may increase our understanding of RNA metabolism during cellular aging.

Membranous glomerulonephritis in a patient with anti-u1 ribonucleoprotein (RNP antibody-positive mixed connective tissue disease: A case report

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Naoya Toriu

2018-03-01

Full Text Available We report a 33-year-old Japanese man diagnosed with mixed connective tissue disease (MCTD who developed nephrotic proteinuria. Both speckled antinuclear antibody (ANA and anti-U1 ribonucleoprotein (RNP antibody were positive, but anti-double-stranded DNA (dsDNA antibody and anti-Smith (Sm antibody were negative, while complement levels were normal. Renal biopsy revealed membranous glomerulonephritis (MGN with diffuse thickening of the glomerular basement membrane (GBM plus spike and bubble formation. Immunofluorescence demonstrated granular deposits of IgG and C3 along the GBM. Analysis of IgG subclasses showed predominant deposition of IgG1 and IgG4, unlike typical lupus nephritis in which there is predominant deposition of IgG1, IgG2, IgG3, and C1q. Electron microscopy identified numerous large electron-dense deposits (EDD of various types in the subepithelial region of the GBM, but there were no EDD localized in the mesangium or subendothelium. Based on these findings, MGN was considered to be closely related to MCTD in this patient.

Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

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Nishimura, Kazuho; Kumazawa, Takuya; Kuroda, Takao; Katagiri, Naohiro; Tsuchiya, Mai; Goto, Natsuka; Furumai, Ryohei; Murayama, Akiko; Yanagisawa, Junn; Kimura, Keiji

2015-03-03

The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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

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

2010-09-24

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. Copyright © 2010 Elsevier Inc. All rights reserved.

Novel monoclonal autoantibody specificity associated with ribonucleoprotein complexes

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Winkler, A.; Watson-McKown, R.; Wise, K.

1986-01-01

The authors describe an IgG/sub 2a/, kappa monoclonal autoantibody (mAb) F78 derived from a 6-month old MRL-Mp lpr/lpr mouse that recognizes a novel epitope associated with small nuclear ribonuclear protein complexes (snRNP). Indirect immunofluorescent staining of HEp-2 cells with F78 showed a nonnucleolar speckled nuclear pattern characteristic of anti-RNP and anti-Sm mAbs which could be abrogated by pretreating fixed cells with 0.1M HCl prior to staining. Immunoblots of whole cell extracts (dissociated in SDS, urea and mercaptan at 4 0 C then subjected to SDS-PAGE) showed that F78 selectively bound to a component of M/sub r/ = 100,000 clearly distinct from components recognized by two mAbs described by Billings et al that detected, respectively, proteins of M/sub r/ = 70,000 associated with RNP and M/sub r/ = 13,000 associated with Sm. Incubation of extracts at 100 0 C prior to SDS-PAGE eliminated subsequent binding of F78 but not of the other nAbs. F78 as well as the other mAbs selectively immunoprecipitated characteristic patterns of small nuclear RNAs (U 1 , U 2 , U 4 , U 5 , U 6 ) from extracts of 32 P-phosphate labeled HeLa cells. These results suggest a new specificity associated with snRNP that is recognized in the MRL autoimmune response

Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

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Han, Juan; Tang, Feng-ming; Pu, Dan; Xu, Dan; Wang, Tao; Li, Weimin

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

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

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

2016-01-01

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. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.

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

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Zhang, Ping [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Wang, Ningling [Department of Assisted Reproduction, Shanghai Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Lin, Xianhua; Jin, Li [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Xu, Hong, E-mail: xuhong1168@126.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Li, Rong [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Huang, Hefeng, E-mail: huanghefg@hotmail.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China)

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. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.

hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons.

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Briese, Michael; Saal-Bauernschubert, Lena; Ji, Changhe; Moradi, Mehri; Ghanawi, Hanaa; Uhl, Michael; Appenzeller, Silke; Backofen, Rolf; Sendtner, Michael

2018-03-20

Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance. Copyright © 2018 the Author(s). Published by PNAS.

ANTI-HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN B1 (ANTI-RA33 ANTIBODIES IN RHEUMATOID ARTHRITIS AND SYSTEMIC SCLEROSIS

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P. A. Kuznetsova

2017-01-01

Full Text Available Anti-heterogeneous nuclear ribonucleoprotein (RNP autoantibodies (AAbs are encountered in many autoimmune rheumatic diseases (ARDs. The potential diagnostic value of the RA33 AAb complex consisting of RNP A2 and alternative domains of the splicing proteins RNP B1 and RNP B2 is now of interest to rheumatologists. Subjects and methods. The authors studied the frequency of anti-RNP B1 AAbs in 300 patients with systemic ARDs, including those with rheumatoid arthritis (RA, ankylosing spondylitis (AS, systemic lupus erythematosus (SLE, systemic sclerosis (SSc, and Sjö gren's syndrome (SS and in 53 people without ARDs, who constituted a control group. Serum anti-RNP B1 AAbs were assessed by enzyme immunoassay. Results and discussion. The frequency of anti-RNP B1 AAbs in patients with ARDs was much higher than that in the control group: 170/300 (56.6% and 8/53 (13% patients, respectively. Anti-RNP B1 AAbs were detected in 78.5% (113/144 of the patients with RA; 40.3% (23/57 of those with AS, in 67.5% (27/40 of those with SSc, in 36.4% (16/44 of those with SLE, and in 13.3% (2/15 of those with SS. The diagnostic sensitivity of the marker for RA was 78.5%, its diagnostic specificity was 84.9%; the likelihood ratio of positive and negative results was 5.24 and 0.24, respectively. In the patients with RA, the level of anti-RNP B1 AAbs significantly correlated with that of C-reactive protein and erythrocyte sedimentation rate, while in those with SSc the detection of anti-RNP B1 AAbs was related to the rigidity of the vascular wall and the presence of hypertension. The frequency of anti-RNP B1 AAbs among the RA patients seronegative for rheumatoid factor and anti-cyclic citrullinated peptide antibodies was 15.4%. Conclusion. Anti-RNP B1 AAs are a useful laboratory marker (with the upper limit of the normal range being 3.3 U/ml, but are of limited value in the diagnosis of RA. Anti-RNP B1 AAbs may be regarded as an additional diagnostic marker for RA.

Functions of Heterogeneous Nuclear Ribonucleoproteins in Stem Cell Potency and Differentiation

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Qishan Chen

2013-01-01

Full Text Available Stem cells possess huge importance in developmental biology, disease modelling, cell replacement therapy, and tissue engineering in regenerative medicine because they have the remarkable potential for self-renewal and to differentiate into almost all the cell types in the human body. Elucidation of molecular mechanisms regulating stem cell potency and differentiation is essential and critical for extensive application. Heterogeneous nuclear ribonucleoproteins (hnRNPs are modular proteins consisting of RNA-binding motifs and auxiliary domains characterized by extensive and divergent functions in nucleic acid metabolism. Multiple roles of hnRNPs in transcriptional and posttranscriptional regulation enable them to be effective gene expression regulators. More recent findings show that hnRNP proteins are crucial factors implicated in maintenance of stem cell self-renewal and pluripotency and cell differentiation. The hnRNPs interact with certain sequences in target gene promoter regions to initiate transcription. In addition, they recognize 3′UTR or 5′UTR of specific gene mRNA forming mRNP complex to regulate mRNA stability and translation. Both of these regulatory pathways lead to modulation of gene expression that is associated with stem cell proliferation, cell cycle control, pluripotency, and committed differentiation.

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

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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.

hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

International Nuclear Information System (INIS)

Berglund, Fredrik M.; Clarke, Paul R.

2009-01-01

Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

Yeast hnRNP-related proteins contribute to the maintenance of telomeres

Energy Technology Data Exchange (ETDEWEB)

Lee-Soety, Julia Y., E-mail: jlee04@sju.edu [Department of Biology, Saint Joseph' s University, PA 19131 (United States); Jones, Jennifer; MacGibeny, Margaret A.; Remaly, Erin C.; Daniels, Lynsey; Ito, Andrea; Jean, Jessica; Radecki, Hannah; Spencer, Shannon [Department of Biology, Saint Joseph' s University, PA 19131 (United States)

2012-09-14

Highlights: Black-Right-Pointing-Pointer Yeast hnRNP-related proteins are able to prevent faster senescence in telomerase-null cells. Black-Right-Pointing-Pointer The conserved RRMs in Npl3 are important for telomere maintenance. Black-Right-Pointing-Pointer Human hnRNP A1 is unable to complement the lack of NPL3 in yeast. Black-Right-Pointing-Pointer Npl3 and Cbc2 may work as telomere capping proteins. -- Abstract: Telomeres protect the ends of linear chromosomes, which if eroded to a critical length can become uncapped and lead to replicative senescence. Telomerase maintains telomere length in some cells, but inappropriate expression facilitates the immortality of cancer cells. Recently, proteins involved in RNA processing and ribosome assembly, such as hnRNP (heterogeneous nuclear ribonucleoprotein) A1, have been found to participate in telomere maintenance in mammals. The Saccharomyces cerevisiae protein Npl3 shares significant amino acid sequence similarities with hnRNP A1. We found that deleting NPL3 accelerated the senescence of telomerase null cells. The highly conserved RNA recognition motifs (RRM) in Npl3 appear to be important for preventing faster senescence. Npl3 preferentially binds telomere sequences in vitro, suggesting that Npl3 may affect telomeres directly. Despite similarities between the two proteins, human hnRNP A1 is unable to complement the lack of Npl3 to rescue accelerated senescence in tlc1 npl3 cells. Deletion of CBC2, which encodes another hnRNP-related protein that associates with Npl3, also accelerates senescence. Potential mechanisms by which hnRNP-related proteins maintain telomeres are discussed.

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin

Science.gov (United States)

Siebrasse, Jan Peter; Veith, Roman; Dobay, Akos; Leonhardt, Heinrich; Daneholt, Bertil; Kubitscheck, Ulrich

2008-01-01

Messenger ribonucleoprotein particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus. To further understand mRNP trafficking, we have studied the intranuclear movement of a specific mRNP, the BR2 mRNP, in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, which allows us to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled with microinjected oligonucleotides (DNA or RNA) complementary to BR2 mRNA or with the RNA-binding protein hrp36, the C. tentans homologue of hnRNP A1. Using high-speed laser microscopy, we followed the intranuclear trajectories of single mRNPs and characterized their motion within the nucleoplasm. The Balbiani ring (BR) mRNPs moved randomly, but unexpectedly, in a discontinuous manner. When mobile, they diffused with a diffusion coefficient corresponding to their size. Between mobile phases, the mRNPs were slowed down 10-to 250-fold but were never completely immobile. Earlier electron microscopy work has indicated that BR particles can attach to thin nonchromatin fibers, which are sometimes connected to discrete fibrogranular clusters. We propose that the observed discontinuous movement reflects transient interactions between freely diffusing BR particles and these submicroscopic structures. PMID:19074261

Epitope mapping of the U1 small nuclear ribonucleoprotein particle in patients with systemic lupus erythematosus and mixed connective tissue disease.

Science.gov (United States)

Somarelli, J A; Mesa, A; Rodriguez, R; Avellan, R; Martinez, L; Zang, Y J; Greidinger, E L; Herrera, R J

2011-03-01

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of 'self ' antigens. Proteins of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein-protein interactions. These data suggest that while some autoantibodies may target U1 snRNP proteins as monomers or apoptosis-induced, protease-digested fragments, others may recognize epitopes on assembled protein subcomplexes of the U1 snRNP. Although nearly all of the peptides are strong predictors of autoimmune illness, none were successful at distinguishing between SLE and MCTD. The antigenicity of some peptides significantly correlated with several clinical symptoms. This investigation implicitly highlights the complexities of autoimmune epitopes, and autoimmune illnesses in general, and demonstrates the variability of antigens in patient populations, all of which contribute to difficult clinical diagnoses.

hnRNP L regulates differences in expression of mouse integrin alpha2beta1.

Science.gov (United States)

Cheli, Yann; Kunicki, Thomas J

2006-06-01

There is a 2-fold variation in platelet integrin alpha2beta1 levels among inbred mouse strains. Decreased alpha2beta1 in 4 strains carrying Itga2 haplotype 2 results from decreased affinity of heterogeneous ribonucleoprotein L (hnRNP L) for a 6 CA repeat sequence (CA6) within intron 1. Seven strains bearing haplotype 1 and a 21 CA repeat sequence at this position (CA21) express twice the level of platelet alpha2beta1 and exhibit an equivalent gain of platelet function in vitro. By UV crosslinking and immunoprecipitation, hnRNP L binds more avidly to CA21, relative to CA6. By cell-free, in vitro mRNA splicing, decreased binding of hnRNP L results in decreased splicing efficiency and an increased proportion of alternatively spliced product. The splicing enhancer activity of CA21 in vivo is abolished by prior treatment with hnRNP L-specific siRNA. Thus, decreased surface alpha2beta1 results from decreased Itga2 pre-mRNA splicing regulated by hnRNP L and depends on CA repeat length at a specific site in intron 1.

hnRNP L regulates differences in expression of mouse integrin α2β1

Science.gov (United States)

Cheli, Yann; Kunicki, Thomas J.

2006-01-01

There is a 2-fold variation in platelet integrin α2β1 levels among inbred mouse strains. Decreased α2β1 in 4 strains carrying Itga2 haplotype 2 results from decreased affinity of heterogeneous ribonucleoprotein L (hnRNP L) for a 6 CA repeat sequence (CA6) within intron 1. Seven strains bearing haplotype 1 and a 21 CA repeat sequence at this position (CA21) express twice the level of platelet α2β1 and exhibit an equivalent gain of platelet function in vitro. By UV crosslinking and immunoprecipitation, hnRNP L binds more avidly to CA21, relative to CA6. By cell-free, in vitro mRNA splicing, decreased binding of hnRNP L results in decreased splicing efficiency and an increased proportion of alternatively spliced product. The splicing enhancer activity of CA21 in vivo is abolished by prior treatment with hnRNP L–specific siRNA. Thus, decreased surface α2β1 results from decreased Itga2 pre-mRNA splicing regulated by hnRNP L and depends on CA repeat length at a specific site in intron 1. PMID:16455949

Specific RNP capture with antisense LNA/DNA mixmers.

Science.gov (United States)

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W

2017-08-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

Energy Technology Data Exchange (ETDEWEB)

Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

2013-01-18

Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins

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Lia Carolina Soares Medeiros

2017-11-01

Full Text Available Trypanosomatids (order Kinetoplastida, including the human pathogens Trypanosoma cruzi (agent of Chagas disease, Trypanosoma brucei, (African sleeping sickness, and Leishmania (leishmaniasis, affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9 technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9, but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9, and in vitro-transcribed single guide RNAs (sgRNAs results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major. RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.

Nuclear surveillance of mRNP formation

DEFF Research Database (Denmark)

Jensen, Torben Heick

Proper formation of mRNP requires co-transcriptional loading of proteins onto nascent transcripts. Mutations in several genes involved in mRNA processing, mRNP assembly and nuclear export lead to production of aberrant mRNPs that are retained in transcription site-associated foci. Retention...... and degradation of transcripts depend on the nuclear exosome of 3’-5’ exonucleases.We have studied connections between mRNP assembly and quality control in the yeast S. cerevisiae using mutants of the THO complex. THO is implicated in co-transcriptional mRNP assembly, but its precise role is not known. Genetic...... and biochemical data now show that a defective THO complex negatively impacts mRNA 3’-end processing. We are currently trying to understand the relationship between this phenomenon and mRNP quality control. Retention of mRNP in THO mutants is dependent on the nuclear exosome component Rrp6p. Using the solved...

Functional organization of the Sm core in the crystal structure of human U1 snRNP.

Science.gov (United States)

Weber, Gert; Trowitzsch, Simon; Kastner, Berthold; Lührmann, Reinhard; Wahl, Markus C

2010-12-15

U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5'-splice site early during spliceosome assembly. It represents a prototype spliceosomal subunit containing a paradigmatic Sm core RNP. The crystal structure of human U1 snRNP obtained from natively purified material by in situ limited proteolysis at 4.4 Å resolution reveals how the seven Sm proteins, each recognize one nucleotide of the Sm site RNA using their Sm1 and Sm2 motifs. Proteins D1 and D2 guide the snRNA into and out of the Sm ring, and proteins F and E mediate a direct interaction between the Sm site termini. Terminal extensions of proteins D1, D2 and B/B', and extended internal loops in D2 and B/B' support a four-way RNA junction and a 3'-terminal stem-loop on opposite sides of the Sm core RNP, respectively. On a higher organizational level, the core RNP presents multiple attachment sites for the U1-specific 70K protein. The intricate, multi-layered interplay of proteins and RNA rationalizes the hierarchical assembly of U snRNPs in vitro and in vivo.

DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication.

Science.gov (United States)

Sui, Jiangdong; Lin, Yu-Fen; Xu, Kangling; Lee, Kyung-Jong; Wang, Dong; Chen, Benjamin P C

2015-07-13

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3' overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3' overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

Energy Technology Data Exchange (ETDEWEB)

Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

2009-11-15

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

The effect of O-GlcNAcylation on hnRNP A1 translocation and interaction with transportin1

Energy Technology Data Exchange (ETDEWEB)

Roth, Shira; Khalaila, Isam, E-mail: isam@bgu.ac.il

2017-01-01

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a major pre-mRNA binding protein involved in transcription and translation. Although predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytosol, delivering its anchored pre-mRNA for further processing. Translocation is important for hnRNP A1 to accomplish its transcriptional and translational roles. Transportin1 (Trn1), a translocation protein, facilitates the translocation of hnRNP A1 back to the nucleus. Moreover, phosphorylation of serine residues at hnRNP A1 C-terminal domain affects its translocation. In this study, we found that phosphorylation is not the only modification that hnRNP A1 undergoes, but also O-linked N-acetylglucosaminylation (O-GlcNAcylation) could occur. Several putative novel O-GlcNAcylation and phosphorylation sites in hnRNP A1 were mapped. Whereas enhanced O-GlcNAcylation increased hnRNP A1 interaction with Trn1, enhanced phosphorylation reduced the interaction between the proteins. In addition, elevated O-GlcNAcylation resulted in hnRNP A1 seclusion in the nucleus, whereas elevated phosphorylation resulted in its accumulation in the cytosol. These findings suggest that a new player, i.e., O-GlcNAcylation, regulates hnRNP A1 translocation and interaction with Trn1, possibly affecting its function. There is a need for further study, to elucidate the role of O-GlcNAcylation in the regulation of the specific activities of hnRNP A1 in transcription and translation. - Highlights: • O-GlcNAcylation regulates hnRNP A1 translocation and interaction with Trn1. • Reciprocity between phosphorylation and O-GlcNAcylation in hnRNP A1 is proposed. • Novel O-GlcNAcylation and phosphorylation sites on hnRNPA1 were identified.

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

International Nuclear Information System (INIS)

Loucaides, Eva M.; Kirchbach, Johann C. von; Foeglein, Agnes; 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.

Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.

Science.gov (United States)

Saliou, Jean-Michel; Manival, Xavier; Tillault, Anne-Sophie; Atmanene, Cédric; Bobo, Claude; Branlant, Christiane; Van Dorsselaer, Alain; Charpentier, Bruno; Cianférani, Sarah

2015-08-01

Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Circular chromatin complexes in human lymphocytes high-resolution autoradiography

International Nuclear Information System (INIS)

Becak, M.L.; Fukuda-Pizzocaro, K.; Santos, R. de C.S. dos; Brunner, O.

1985-01-01

Transcriptionally active chromatin fibers were observed in chromosomes presenting the loops/scaffold configuration. The active fibers showed altered nucleosomes and presented multiforked aspects which led to the formation of ring complexes. The ribonucleoprotein transcripts (RNP) appeared as networks of 0.1 μm or multiples tandemly disposed along the fiber. It is suggested that the ring complexes belong to the human genome. The possibility that these circular structures come from a prokaryote is also considered. (author) [pt

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes.

Science.gov (United States)

Meccariello, Angela; Monti, Simona Maria; Romanelli, Alessandra; Colonna, Rita; Primo, Pasquale; Inghilterra, Maria Grazia; Del Corsano, Giuseppe; Ramaglia, Antonio; Iazzetti, Giovanni; Chiarore, Antonia; Patti, Francesco; Heinze, Svenia D; Salvemini, Marco; Lindsay, Helen; Chiavacci, Elena; Burger, Alexa; Robinson, Mark D; Mosimann, Christian; Bopp, Daniel; Saccone, Giuseppe

2017-08-30

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests.

Science.gov (United States)

Gillet, François-Xavier; Garcia, Rayssa A; Macedo, Leonardo L P; Albuquerque, Erika V S; Silva, Maria C M; Grossi-de-Sa, Maria F

2017-01-01

Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil ( Anthonomus grandis ), we showed that the chimeric protein PTD-DRBD (peptide transduction domain-dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests

Directory of Open Access Journals (Sweden)

François-Xavier Gillet

2017-04-01

Full Text Available Genetically modified (GM crops producing double-stranded RNAs (dsRNAs are being investigated largely as an RNA interference (RNAi-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis, we showed that the chimeric protein PTD-DRBD (peptide transduction domain—dsRNA binding domain combined with dsRNA forms a ribonucleoprotein particle (RNP that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

Regulatory RNPs: a novel class of ribonucleoproteins that potentially contribute to ribosome heterogeneity

Directory of Open Access Journals (Sweden)

Aaron R. Poole

2017-09-01

Full Text Available Many ribonucleoproteins (RNPs, which are comprised of noncoding RNA and associated proteins, are involved in essential cellular processes such as translation and pre-mRNA splicing. One class of RNP is the small Cajal body-specific RNP (scaRNP, which contributes to the biogenesis of small nuclear RNPs (snRNPs that are central components of the spliceosome. Three scaRNAs are internally processed, generating stable nucleolus-enriched RNAs of unknown function. Here, we provide data that show that these RNAs become part of RNPs we term regulatory RNPs (regRNPs. Most modifications within rRNA (predominantly pseudouridylation and ribose 2′-O-methylation are conducted by small nucleolar RNPs (snoRNPs, and we provide evidence that the activity of at least some of these snoRNPs is under the control of regRNPs. Because modifications within rRNA can vary in different physiological or pathological situations, rRNA modifications are thought to be the major source of ribosome heterogeneity. Our identification of regRNPs thus provides a potential mechanism for how ribosome heterogeneity may be accomplished. This work also provides additional functional connections between the Cajal body and the nucleolus.

Torsin Mediates Primary Envelopment of Large Ribonucleoprotein Granules at the Nuclear Envelope

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Vahbiz Jokhi

2013-04-01

Full Text Available A previously unrecognized mechanism through which large ribonucleoprotein (megaRNP granules exit the nucleus is by budding through the nuclear envelope (NE. This mechanism is akin to the nuclear egress of herpes-type viruses and is essential for proper synapse development. However, the molecular machinery required to remodel the NE during this process is unknown. Here, we identify Torsin, an AAA-ATPase that in humans is linked to dystonia, as a major mediator of primary megaRNP envelopment during NE budding. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the messenger RNAs contained within fail to reach synaptic sites, preventing normal synaptic protein synthesis and thus proper synaptic bouton development. These studies begin to establish the cellular machinery underlying the exit of megaRNPs via budding, offer an explanation for the “nuclear blebbing” phenotype found in dystonia models, and provide an important link between Torsin and the synaptic phenotypes observed in dystonia.

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles.

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Wieben, E D; Nenninger, J M; Pederson, T

1985-05-05

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

Matrix metalloproteinase 12 is induced by heterogeneous nuclear ribonucleoprotein K and promotes migration and invasion in nasopharyngeal carcinoma

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Chung, I-Che; Li, Hsin-Pai; Chang, Yu-Sun; Chen, Lih-Chyang; Chung, An-Ko; Chao, Mei; Huang, Hsin-Yi; Hsueh, Chuen; Tsang, Ngan-Ming; Chang, Kai-Ping; Liang, Ying

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a DNA/RNA binding protein, is associated with metastasis in nasopharyngeal carcinoma (NPC). However, the mechanisms underlying hnRNP K-mediated metastasis is unclear. The aim of the present study was to determine the role of matrix metalloproteinase (MMP) in hnRNP K-mediated metastasis in NPC. We studied hnRNP K-regulated MMPs by analyzing the expression profiles of MMP family genes in NPC tissues and hnRNP K-knockdown NPC cells using Affymetrix microarray analysis and quantitative RT-PCR. The association of hnRNP K and MMP12 expression in 82 clinically proven NPC cases was determined by immunohistochemical analysis. The hnRNP K-mediated MMP12 regulation was determined by zymography and Western blot, as well as by promoter, DNA pull-down and chromatin immunoprecipitation (ChIP) assays. The functional role of MMP12 in cell migration and invasion was demonstrated by MMP12-knockdown and the treatment of MMP12-specific inhibitor, PF-356231. MMP12 was overexpressed in NPC tissues, and this high level of expression was significantly correlated with high-level expression of hnRNP K (P = 0.026). The levels of mRNA, protein and enzyme activity of MMP12 were reduced in hnRNP K-knockdown NPC cells. HnRNP K interacting with the region spanning −42 to −33 bp of the transcription start site triggered transcriptional activation of the MMP12 promoter. Furthermore, inhibiting MMP12 by MMP12 knockdown and MMP12-specific inhibitor, PF-356231, significantly reduced the migration and invasion of NPC cells. Overexpression of MMP12 was significantly correlated with hnRNP K in NPC tissues. HnRNP K can induce MMP12 expression and enzyme activity through activating MMP12 promoter, which promotes cell migration and invasion in NPC cells. In vitro experiments suggest that NPC metastasis with high MMP12 expression may be treated with PF-356231. HnRNP K and MMP12 may be potential therapeutic markers for NPC, but

Spinal muscular atrophy: Selective motor neuron loss and global defect in the assembly of ribonucleoproteins.

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Beattie, Christine E; Kolb, Stephen J

2018-08-15

Spinal muscular atrophy is caused by deletions or mutations in the SMN1 gene that result in reduced expression of the SMN protein. The SMN protein is an essential molecular chaperone that is required for the biogenesis of multiple ribonucleoprotein (RNP) complexes including spliceosomal small nuclear RNPs (snRNPs). Reductions in SMN expression result in a reduced abundance of snRNPs and to downstream RNA splicing alterations. SMN is also present in axons and dendrites and appears to have important roles in the formation of neuronal mRNA-protein complexes during development or neuronal repair. Thus, SMA is an exemplar, selective motor neuron disorder that is caused by defects in fundamental RNA processing events. A detailed molecular understanding of how motor neurons fail, and why other neurons do not, in SMA will yield important principals about motor neuron maintenance and neuronal specificity in neurodegenerative diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression of anti-heterogenous nuclear ribonucleoprotein (anti-hnRNP in limited systemic sclerosis patients: Relation to radiographic findings of the hand

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Nihal Fathi

2018-01-01

Conclusion: Joint affection in SSc is more frequent than expected. Anti-hnRNP A1 and anti hnRNP A2 antigens may be useful markers for SSc patient although no significant relation was found with radiologic findings.

The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

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Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

2014-07-18

In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles.

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Mout, Rubul; Rotello, Vincent M

2017-10-20

In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users' need. Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al ., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al ., 2017). We report here only the generation of the delivery vehicle i.e. , the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

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Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

2016-08-15

Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. © 2016 The Author(s).

Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex.

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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; Wright, Elizabeth R

2014-07-01

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. 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 organizational profiles

Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity

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Cédric Schelcher

2016-06-01

Full Text Available RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

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Theuser, Matthias; Höbartner, Claudia; Wahl, Markus C.; Santos, Karine F.

2016-01-01

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA–protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing. PMID:27354531

The 7SK snRNP associates with the little elongation complex to promote snRNA gene expression.

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Egloff, Sylvain; Vitali, Patrice; Tellier, Michael; Raffel, Raoul; Murphy, Shona; Kiss, Tamás

2017-04-03

The 7SK small nuclear RNP (snRNP), composed of the 7SK small nuclear RNA (snRNA), MePCE, and Larp7, regulates the mRNA elongation capacity of RNA polymerase II (RNAPII) through controlling the nuclear activity of positive transcription elongation factor b (P-TEFb). Here, we demonstrate that the human 7SK snRNP also functions as a canonical transcription factor that, in collaboration with the little elongation complex (LEC) comprising ELL, Ice1, Ice2, and ZC3H8, promotes transcription of RNAPII-specific spliceosomal snRNA and small nucleolar RNA (snoRNA) genes. The 7SK snRNA specifically associates with a fraction of RNAPII hyperphosphorylated at Ser5 and Ser7, which is a hallmark of RNAPII engaged in snRNA synthesis. Chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) experiments revealed enrichments for all components of the 7SK snRNP on RNAPII-specific sn/snoRNA genes. Depletion of 7SK snRNA or Larp7 disrupts LEC integrity, inhibits RNAPII recruitment to RNAPII-specific sn/snoRNA genes, and reduces nascent snRNA and snoRNA synthesis. Thus, through controlling both mRNA elongation and sn/snoRNA synthesis, the 7SK snRNP is a key regulator of nuclear RNA production by RNAPII. © 2017 The Authors.

Alterations in polyribosome and messenger ribonucleic acid metabolism and messenger ribonucleoprotein utilization in osmotically stressed plant seedlings

International Nuclear Information System (INIS)

Mason, H.S.

1986-01-01

Polyribosome aggregation state in growing tissues of barley and wheat leaf of stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf of stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of 32 PO 4 3- into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A) + RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A) + RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described

Genome-wide analysis of KAP1, the 7SK snRNP complex, and RNA polymerase II

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Ryan P. McNamara

2016-03-01

Full Text Available The transition of RNA polymerase II (Pol II from transcription initiation into productive elongation in eukaryotic cells is regulated by the P-TEFb kinase, which phosphorylates the C-terminal domain of paused Pol II at promoter-proximal regions. Our recent study found that P-TEFb (in an inhibited state bound to the 7SK snRNP complex interacts with the KAP1/TRIM28 transcriptional regulator, and that KAP1 and the 7SK snRNP co-occupy most gene promoters containing paused Pol II. Here we provide a detailed experimental description and analysis of the ChIP-seq datasets that have been deposited into Gene Expression Omnibus (GEO: GS72622, so that independent groups can replicate and expand upon these findings. We propose these datasets would provide valuable information for researchers studying mechanisms of transcriptional regulation including Pol II pausing and pause release. Keywords: P-TEFb/7SK snRNP, KAP1, RNA polymerase II, ChIP-seq, Transcription elongation

RNA and RNP as Building Blocks for Nanotechnology and Synthetic Biology.

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Ohno, Hirohisa; Saito, Hirohide

2016-01-01

Recent technologies that aimed to elucidate cellular function have revealed essential roles for RNA molecules in living systems. Our knowledge concerning functional and structural information of naturally occurring RNA and RNA-protein (RNP) complexes is increasing rapidly. RNA and RNP interaction motifs are structural units that function as building blocks to constitute variety of complex structures. RNA-central synthetic biology and nanotechnology are constructive approaches that employ the accumulated information and build synthetic RNA (RNP)-based circuits and nanostructures. Here, we describe how to design and construct synthetic RNA (RNP)-based devices and structures at the nanometer-scale for biological and future therapeutic applications. RNA/RNP nanostructures can also be utilized as the molecular scaffold to control the localization or interactions of target molecule(s). Moreover, RNA motifs recognized by RNA-binding proteins can be applied to make protein-responsive translational "switches" that can turn gene expression "on" or "off" depending on the intracellular environment. This "synthetic RNA and RNP world" will expand tools for nanotechnology and synthetic biology. In addition, these reconstructive approaches would lead to a greater understanding of building principle in naturally occurring RNA/RNP molecules and systems. Copyright © 2016 Elsevier Inc. All rights reserved.

Cytoplasmic assembly of snRNP particles from stored proteins and newly transcribed snRNA's in L929 mouse fibroblasts

International Nuclear Information System (INIS)

Sauterer, R.A.; Feeney, R.J.; Zieve, G.W.

1988-01-01

Newly synthesized snRNAs appear transiently in the cytoplasm where they assemble into ribonucleoprotein particles, the snRNP particles, before returning permanently to the interphase nucleus. In this report, bona fide cytoplasmic fractions, prepared by cell enucleation, are used for a quantitative analysis of snRNP assembly in growing mouse fibroblasts. The half-lives and abundances of the snRNP precursors in the cytoplasm and the rates of snRNP assembly are calculated in L929 cells. With the exception of U6, the major snRNAs are stable RNA species; U1 is almost totally stable while U2 has a half-life of about two cell cycles. In contrast, the majority of newly synthesized U6 decays with a half-life of about 15 h. The relative abundances of the newly synthesized snRNA species U1, U2, U3, U4 and U6 in the cytoplasm are determined by Northern hybridization using cloned probes and are approximately 2% of their nuclear abundance. The half-lives of the two major snRNA precursors in the cytoplasm (U1 and U2) are approximately 20 min as determined by labeling to steady state. The relative abundance of the snRNP B protein in the cytoplasm is determined by Western blotting with the Sm class of autoantibodies and is approximately 25% of the nuclear abundance. Kinetic studies, using the Sm antiserum to immunoprecipitate the methionine-labeled snRNP proteins, suggest that the B protein has a half-life of 90 to 120 min in the cytoplasm. These data are discussed and suggest that there is a large pool of more stable snRNP proteins in the cytoplasm available for assembly with the less abundant but more rapidly turning-over snRNAs

Induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway delays the initiation but fails to eradicate established murine acute myeloid leukemia.

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Jaako, P; Ugale, A; Wahlestedt, M; Velasco-Hernandez, T; Cammenga, J; Lindström, M S; Bryder, D

2017-01-01

Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

The role of polypyrimidine tract-binding proteins and other hnRNP proteins in plant splicing regulation

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Andreas eWachter

2012-05-01

Full Text Available Alternative precursor mRNA splicing is a widespread phenomenon in multicellular eukaryotes and represents a major means for functional expansion of the transcriptome. While several recent studies have revealed an important link between splicing regulation and fundamental biological processes in plants, many important aspects, such as the underlying splicing regulatory mechanisms, are so far not well understood. Splicing decisions are in general based on a splicing code that is determined by the dynamic interplay of splicing-controlling factors and cis-regulatory elements. Several members of the group of heterogeneous nuclear ribonucleoprotein (hnRNP proteins are well-known regulators of splicing in animals and the comparatively few reports on some of their plant homologues revealed similar functions. This also applies to polypyrimidine tract-binding proteins (PTBs, a thoroughly investigated class of hnRNP proteins with splicing regulatory functions in both animals and plants. Further examples from plants are auto- and cross-regulatory splicing circuits of glycine-rich RNA-binding proteins (GRPs and splicing enhancement by oligouridylatebinding proteins. Besides their role in defining splice site choice, hnRNP proteins are also involved in multiple other steps of nucleic acid metabolism, highlighting the functional versatility of this group of proteins in higher eukaryotes.

U1 snRNP Alteration and Neuronal Cell Cycle Reentry in Alzheimer Disease

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Bing Bai

2018-03-01

Full Text Available The aberrancy of U1 small nuclear ribonucleoprotein (snRNP complex and RNA splicing has been demonstrated in Alzheimer’s disease (AD. Importantly, the U1 proteopathy is AD-specific, widespread and early-occurring, thus providing a very unique clue to the AD pathogenesis. The prominent feature of U1 histopathology is its nuclear depletion and redistribution in the neuronal cytoplasm. According to the preliminary data, the initial U1 cytoplasmic distribution pattern is similar to the subcellular translocation of the spliceosome in cells undergoing mitosis. This implies that the U1 mislocalization might reflect the neuronal cell cycle-reentry (CCR which has been extensively evidenced in AD brains. The CCR phenomenon explains the major molecular and cellular events in AD brains, such as Tau and amyloid precursor protein (APP phosphorylation, and the possible neuronal death through mitotic catastrophe (MC. Furthermore, the CCR might be mechanistically linked to inflammation, a critical factor in the AD etiology according to the genetic evidence. Therefore, the discovery of U1 aberrancy might strengthen the involvement of CCR in the AD neuronal degeneration.

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

International Nuclear Information System (INIS)

Quaresma, Alexandre J.C.; Bressan, G.C.; Gava, L.M.; Lanza, D.C.F.; Ramos, C.H.I; Kobarg, Joerg

2009-01-01

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182 - GWbs marker protein and TIA-1 stress granule component

CryoEM structures of two spliceosomal complexes: starter and dessert at the spliceosome feast.

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Nguyen, Thi Hoang Duong; Galej, Wojciech P; Fica, Sebastian M; Lin, Pei-Chun; Newman, Andrew J; Nagai, Kiyoshi

2016-02-01

The spliceosome is formed on pre-mRNA substrates from five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs), and numerous non-snRNP factors. Saccharomyces cerevisiae U4/U6.U5 tri-snRNP comprises U5 snRNA, U4/U6 snRNA duplex and approximately 30 proteins and represents a substantial part of the spliceosome before activation. Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex is a post-catalytic intron lariat spliceosome containing U2 and U5 snRNPs, NTC (nineteen complex), NTC-related proteins (NTR), U6 snRNA, and an RNA intron lariat. Two recent papers describe near-complete atomic structures of these complexes based on cryoEM single-particle analysis. The U4/U6.U5 tri-snRNP structure provides crucial insight into the activation mechanism of the spliceosome. The U2.U6.U5 complex reveals the striking architecture of NTC and NTR and important features of the group II intron-like catalytic RNA core remaining after spliced mRNA is released. These two structures greatly advance our understanding of the mechanism of pre-mRNA splicing. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1

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Gao, Guozhen; Dhar, Surbhi

2017-01-01

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

Selective incorporation of vRNP into influenza A virions determined by its specific interaction with M1 protein

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Chaimayo, Chutikarn [Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642 (United States); Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Hayashi, Tsuyoshi; Underwood, Andrew; Hodges, Erin [Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642 (United States); Takimoto, Toru, E-mail: toru_takimoto@urmc.rochester.edu [Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642 (United States)

2017-05-15

Influenza A viruses contain eight single-stranded, negative-sense RNA segments as viral genomes in the form of viral ribonucleoproteins (vRNPs). During genome replication in the nucleus, positive-sense complementary RNPs (cRNPs) are produced as replicative intermediates, which are not incorporated into progeny virions. To analyze the mechanism of selective vRNP incorporation into progeny virions, we quantified vRNPs and cRNPs in the nuclear and cytosolic fractions of infected cells, using a strand-specific qRT-PCR. Unexpectedly, we found that cRNPs were also exported to the cytoplasm. This export was chromosome region maintenance 1 (CRM1)-independent unlike that of vRNPs. Although both vRNPs and cRNPs were present in the cytosol, viral matrix (M1) protein, a key regulator for viral assembly, preferentially bound vRNPs over cRNPs. These results indicate that influenza A viruses selectively uptake cytosolic vRNPs through a specific interaction with M1 during viral assembly. - Highlights: •Influenza cRNPs are exported from the nucleus of an infected cell via a CRM1-independent pathway. •Influenza A viruses selectively incorporate cytosolic vRNPs through a specific interaction with M1 during viral assembly. •M1 dissociates from vRNP export complex after nuclear export, and is re-associated with vRNPs at the plasma membrane.

Selective incorporation of vRNP into influenza A virions determined by its specific interaction with M1 protein

International Nuclear Information System (INIS)

Chaimayo, Chutikarn; Hayashi, Tsuyoshi; Underwood, Andrew; Hodges, Erin; Takimoto, Toru

2017-01-01

Influenza A viruses contain eight single-stranded, negative-sense RNA segments as viral genomes in the form of viral ribonucleoproteins (vRNPs). During genome replication in the nucleus, positive-sense complementary RNPs (cRNPs) are produced as replicative intermediates, which are not incorporated into progeny virions. To analyze the mechanism of selective vRNP incorporation into progeny virions, we quantified vRNPs and cRNPs in the nuclear and cytosolic fractions of infected cells, using a strand-specific qRT-PCR. Unexpectedly, we found that cRNPs were also exported to the cytoplasm. This export was chromosome region maintenance 1 (CRM1)-independent unlike that of vRNPs. Although both vRNPs and cRNPs were present in the cytosol, viral matrix (M1) protein, a key regulator for viral assembly, preferentially bound vRNPs over cRNPs. These results indicate that influenza A viruses selectively uptake cytosolic vRNPs through a specific interaction with M1 during viral assembly. - Highlights: •Influenza cRNPs are exported from the nucleus of an infected cell via a CRM1-independent pathway. •Influenza A viruses selectively incorporate cytosolic vRNPs through a specific interaction with M1 during viral assembly. •M1 dissociates from vRNP export complex after nuclear export, and is re-associated with vRNPs at the plasma membrane.

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

Science.gov (United States)

Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

2016-03-15

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

The human cap-binding complex is functionally connected to the nuclear RNA exosome

DEFF Research Database (Denmark)

Andersen, Peter Refsing; Domanski, Michal; Kristiansen, Maiken Søndergaard

2013-01-01

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome...... and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis...

Up-regulation and subcellular localization of hnRNP A2/B1 in the development of hepatocellular carcinoma

International Nuclear Information System (INIS)

Cui, Huaqing; Wu, Feng; Sun, Yanling; Fan, Guocai; Wang, Qingming

2010-01-01

Hepatocellular carcinoma (HCC) is one of the world's leading causes of death among cancer patients. It is important to find a new biomarker that diagnoses HCC and monitors its treatment. In our previous work, we screened a single-chain antibody (scFv) N14, which could specifically recognize human HepG2 HCC cells but not human non-cancerous liver LO2 cells. However, the antigen it recognized in the cells remained unknown. Recombinant scFv N14 antibody was expressed as an active antibody. Using this antibody with a combination of immunological and proteomic approaches, we identified the antigen of scFv N14 antibody as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). The expression of hnRNP A2/B1 in HCC cells was then investigated by semi-quantitative RT-PCR and immunohistochemistry. We found that the up-regulation of hnRNP A2/B1 was measured at both transcriptional and translational levels in rat HCC cells but not in rat hepatic cells. We also found that in various human hepatic tissues, hnRNP A2/B1 was highly expressed in both human hepatitis virus positive liver tissues and human HCC tissues but not in normal liver tissues. Interestingly, we observed that the localization of hnRNP A2/B1 in HCC cells was altered during the development of HCC. In human hepatitis virus infected tissues hnRNP A2/B1 resides exclusively in the nuclei of hepatocytes. However, when the HCC progressed from a well differentiated to a poorly differentiated stage, hnRNP A2/B1 was increasingly localized in the cytoplasm. In contrast, the HCC tissues with hnRNP A2/B1 highly expressed in the nucleus decreased. This work is the first to show that hnRNP A2/B1 is the antigen specifically recognized by the scFv N14 antibody in HCC cells. The over-expression of hnRNP A2/B1 was confirmed in cultured human and rat HCC cell lines, human virus related hepatitis liver tissues and human HCC tissues. The increased localization of hnRNP A2/B1 in the cytoplasm of HCC cells was revealed

Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

Science.gov (United States)

Jaako, P; Debnath, S; Olsson, K; Zhang, Y; Flygare, J; Lindström, M S; Bryder, D; Karlsson, S

2015-11-01

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-β1-induced kidney injury in a mouse model of diabetes.

Science.gov (United States)

Lo, Chao-Sheng; Shi, Yixuan; Chang, Shiao-Ying; Abdo, Shaaban; Chenier, Isabelle; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao-Ling; Chan, John S D

2015-10-01

We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. Adult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. Overexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1-7 receptor [MasR]) expression, and normalised urinary Ang 1-7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression. These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

Science.gov (United States)

Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C; Gangopadhyay, Jaya; Borufka, Carl; Gygi, Steven P; Gao, Fen-Biao; Reed, Robin

2017-06-13

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients

Directory of Open Access Journals (Sweden)

Shanye Yin

2017-06-01

Full Text Available Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR and glycine-arginine (GR toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.

Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

Science.gov (United States)

Hardin, John W.; Warnasooriya, Chandani; Kondo, Yasushi; Nagai, Kiyoshi; Rueda, David

2015-01-01

In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31. PMID:26503251

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

Science.gov (United States)

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. Copyright © 2014 Elsevier B.V. All rights reserved.

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins

International Nuclear Information System (INIS)

Hoffman, D.W.; Query, C.C.; Golden, B.L.; White, S.W.; Keene, J.D.

1991-01-01

An RNA recognition motif (RRM) of ∼80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel β-strands and two α-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent β-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface to the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins

hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

Energy Technology Data Exchange (ETDEWEB)

Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

2014-01-20

The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

A natural component from Euphorbia humifusa Willd displays novel, broad-spectrum anti-influenza activity by blocking nuclear export of viral ribonucleoprotein

Energy Technology Data Exchange (ETDEWEB)

Chang, So Young; Park, Ji Hoon [Respiratory Viruses Research Laboratory, Discovery Biology Department, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 Beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400 (Korea, Republic of); Kim, Young Ho; Kang, Jong Seong [College of Pharmacy, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Min, Ji-Young, E-mail: jiyoung.min@ip-korea.org [Respiratory Viruses Research Laboratory, Discovery Biology Department, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 Beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400 (Korea, Republic of)

2016-03-04

The need to develop anti-influenza drugs with novel antiviral mechanisms is urgent because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. We identified a novel anti-influenza molecule by screening 861 plant-derived natural components using a high-throughput image-based assay that measures inhibition of the influenza virus infection. 1,3,4,6-tetra-O-galloyl-β-D-glucopyranoside (TGBG) from Euphorbia humifusa Willd showed broad-spectrum anti-influenza activity against two seasonal influenza A strains, A/California/07/2009 (H1N1) and A/Perth/16/2009 (H3N2), and seasonal influenza B strain B/Florida/04/2006. We investigated the mode of action of TGBG using neuraminidase activity inhibition and time-of-addition assays, which evaluate the viral release and entry steps, respectively. We found that TGBG exhibits a novel antiviral mechanism that differs from the FDA-approved anti-influenza drugs oseltamivir which inhibits viral release, and amantadine which inhibits viral entry. Immunofluorescence assay demonstrated that TGBG significantly inhibits nuclear export of influenza nucleoproteins (NP) during the early stages of infection causing NP to accumulate in the nucleus. In addition, influenza-induced activation of the Akt signaling pathway was suppressed by TGBG in a dose-dependent manner. These data suggest that a putative mode of action of TGBG involves inhibition of viral ribonucleoprotein (vRNP) export from the nucleus to the cytoplasm consequently disrupting the assembly of progeny virions. In summary, TGBG has potential as novel anti-influenza therapeutic with a novel mechanism of action. - Highlights: • The plant-derived natural product TGBG has broad-spectrum antiviral activity against seasonal influenza A and B viruses. • TGBG has a novel anti-viral mechanism of action that from differs from the currently available anti-influenza drugs. • TGBG hinders nuclear export of the influenza virus ribonucleoprotein (vRNP

A natural component from Euphorbia humifusa Willd displays novel, broad-spectrum anti-influenza activity by blocking nuclear export of viral ribonucleoprotein

International Nuclear Information System (INIS)

Chang, So Young; Park, Ji Hoon; Kim, Young Ho; Kang, Jong Seong; Min, Ji-Young

2016-01-01

The need to develop anti-influenza drugs with novel antiviral mechanisms is urgent because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. We identified a novel anti-influenza molecule by screening 861 plant-derived natural components using a high-throughput image-based assay that measures inhibition of the influenza virus infection. 1,3,4,6-tetra-O-galloyl-β-D-glucopyranoside (TGBG) from Euphorbia humifusa Willd showed broad-spectrum anti-influenza activity against two seasonal influenza A strains, A/California/07/2009 (H1N1) and A/Perth/16/2009 (H3N2), and seasonal influenza B strain B/Florida/04/2006. We investigated the mode of action of TGBG using neuraminidase activity inhibition and time-of-addition assays, which evaluate the viral release and entry steps, respectively. We found that TGBG exhibits a novel antiviral mechanism that differs from the FDA-approved anti-influenza drugs oseltamivir which inhibits viral release, and amantadine which inhibits viral entry. Immunofluorescence assay demonstrated that TGBG significantly inhibits nuclear export of influenza nucleoproteins (NP) during the early stages of infection causing NP to accumulate in the nucleus. In addition, influenza-induced activation of the Akt signaling pathway was suppressed by TGBG in a dose-dependent manner. These data suggest that a putative mode of action of TGBG involves inhibition of viral ribonucleoprotein (vRNP) export from the nucleus to the cytoplasm consequently disrupting the assembly of progeny virions. In summary, TGBG has potential as novel anti-influenza therapeutic with a novel mechanism of action. - Highlights: • The plant-derived natural product TGBG has broad-spectrum antiviral activity against seasonal influenza A and B viruses. • TGBG has a novel anti-viral mechanism of action that from differs from the currently available anti-influenza drugs. • TGBG hinders nuclear export of the influenza virus ribonucleoprotein (vRNP

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

International Nuclear Information System (INIS)

Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

2008-01-01

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells

Pumping RNA: nuclear bodybuilding along the RNP pipeline.

Science.gov (United States)

Matera, A Gregory; Shpargel, Karl B

2006-06-01

Cajal bodies (CBs) are nuclear subdomains involved in the biogenesis of several classes of small ribonucleoproteins (RNPs). A number of recent advances highlight progress in the understanding of the organization and dynamics of CB components. For example, a class of small Cajal body-specific (sca) RNPs has been discovered. Localization of scaRNPs to CBs was shown to depend on a conserved RNA motif. Intriguingly, this motif is also present in mammalian telomerase RNA and the evidence suggests that assembly of the active form of telomerase RNP occurs in and around CBs during S phase. Important steps in the assembly and modification of spliceosomal RNPs have also been shown to take place in CBs. Additional experiments have revealed the existence of kinetically distinct subclasses of CB components. Finally, the recent identification of novel markers for CBs in both Drosophila and Arabidopsis not only lays to rest questions about the evolutionary conservation of these nuclear suborganelles, but also should enable forward genetic screens for the identification of new components and pathways involved in their assembly, maintenance and function.

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

Science.gov (United States)

Li, Ping; Noegel, Angelika A

2015-11-16

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The ribonucleoprotein Csr network.

Science.gov (United States)

Seyll, Ethel; Van Melderen, Laurence

2013-11-08

Ribonucleoprotein complexes are essential regulatory components in bacteria. In this review, we focus on the carbon storage regulator (Csr) network, which is well conserved in the bacterial world. This regulatory network is composed of the CsrA master regulator, its targets and regulators. CsrA binds to mRNA targets and regulates translation either negatively or positively. Binding to small non-coding RNAs controls activity of this protein. Expression of these regulators is tightly regulated at the level of transcription and stability by various global regulators (RNAses, two-component systems, alarmone). We discuss the implications of these complex regulations in bacterial adaptation.

CRISPR/Cas9 mediated high efficiency knockout of the eye color gene vermillion in Helicoverpa zea (Boddie)

Science.gov (United States)

Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consists of ribonucleoprotein (RNP) complexes formed by combining guide RNA...

The Ribonucleoprotein Csr Network

Directory of Open Access Journals (Sweden)

Ethel Seyll

2013-11-01

Full Text Available Ribonucleoprotein complexes are essential regulatory components in bacteria. In this review, we focus on the carbon storage regulator (Csr network, which is well conserved in the bacterial world. This regulatory network is composed of the CsrA master regulator, its targets and regulators. CsrA binds to mRNA targets and regulates translation either negatively or positively. Binding to small non-coding RNAs controls activity of this protein. Expression of these regulators is tightly regulated at the level of transcription and stability by various global regulators (RNAses, two-component systems, alarmone. We discuss the implications of these complex regulations in bacterial adaptation.

Detection of serum anti-B/B’ UsnRNP antibodies in patients with connective tissue diseases by immunoblotting

Directory of Open Access Journals (Sweden)

L. Iaccarino

2011-09-01

Full Text Available Objective: To investigate the reliability of the immunoblot method in the detection of serum immunoreactivity towards the B/B’ polypeptides of U small nuclear ribonucleoproteins (UsnRNP and to assess the significance of these antibodies in connective tissue disease (CTD patients. Methods: We tested the sera of 348 patients with CTD (101 SLE, 51 systemic sclerosis, 53 primary Sjogren’s syndrome, 27 poly/dermatomyositis, 15 rheumatoid arthritis and 101 overlap CTD, of 31 matched healthy subjects and 13 patients with primary Epstein-Barr virus (EBV infection with high titre IgG anti-EBV antibodies. IgG anti-UsnRNP antibodies were determined by immunoblotting on nuclear extract from Raji cells (an EBV-immortalised human B lymphoid cell line and Jurkat cells (a human T lymphoid cell line. Anti-dsDNA antibodies were detected by indirect immunofluorescence on Crithidia luciliae and anti-ENA by counterimmunoelectrophoresis. Anti-dsDNA activity and avidity were measured in SLE sera by ELISA with Scatchard analysis. Results were statistically analysed by chi-square and Mann-Whitney tests. Results: A high frequency of anti-B/B’ antibodies was found in the sera of CTD patients, confined to SLE (54.4% and overlap CTD with SLE features (55,2%. Anti-B/B’ immune reactivity was closely associated with other anti-UsnRNP specificities, gel precipitating anti-nRNP and anti-P antibodies. Nine out of 15 (60% anti-B/B’ positive/anti-ENA negative lupus sera on Raji blots were confirmed to be positive also on Jurkat blots. The sera from patients with EBV infection provided, on Raji blots, completely different band patterns from those obtained with auto-immune sera. Conclusions. The Sm B/B’ proteins are the predominant or, at least, the most frequently targeted antigens of the UsnRNP auto-immune response in SLE and “lupus-like” overlap CTD. Moreover, anti-B/B’ is diagnostically specific for CTD with SLE features. Immunoblotting on human B lymphoid cells

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability.

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Robart, Aaron R; O'Connor, Catherine M; Collins, Kathleen

2010-03-01

Telomerase adds simple-sequence repeats to chromosome 3' ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.

High-Frequency Promoter Firing Links THO Complex Function to Heavy Chromatin Formation

DEFF Research Database (Denmark)

Mouaikel, John; Causse, Sébastien Z; Rougemaille, Mathieu

2013-01-01

The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes...... (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single......-molecule fluorescence insitu hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase...

Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site.

Science.gov (United States)

Calviño, Fabiola R; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

2015-04-07

During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

A glimpse at mRNA dynamics reveals cellular domains and rapid trafficking through granules

NARCIS (Netherlands)

Gemert, Alice Myriam Christi van

2011-01-01

mRNA transport and targeting are essential to gene expression regulation. Specific mRNA sequences can bind several proteins and together form RiboNucleoProtein particles (RNP). The various proteins within the RNP determine mRNA fate: translation, transport or decay. RNP composition varies with

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Science.gov (United States)

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. © 2016. Published by The Company of Biologists Ltd.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes

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Caroline Vindry

2017-08-01

Full Text Available Pat1 RNA-binding proteins, enriched in processing bodies (P bodies, are key players in cytoplasmic 5′ to 3′ mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA. Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3′ UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors

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Estruch Francisco

2012-09-01

Full Text Available Abstract Background The various steps of mRNP biogenesis (transcription, processing and export are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. Results We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC formation. Conclusions Our results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.

Molecular insights into the specific recognition between the RNA binding domain qRRM2 of hnRNP F and G-tract RNA: A molecular dynamics study.

Science.gov (United States)

Wang, Lingyun; Yan, Feng

2017-12-09

Heterogeneous nuclear ribonucleoprotein F (hnRNP F) controls the expression of various genes through regulating the alternative splicing of pre-mRNAs in the nucleus. It uses three quasi-RNA recognition motifs (qRRMs) to recognize G-tract RNA which contains at least three consecutive guanines. The structures containing qRRMs of hnRNP F in complex with G-tract RNA have been determined by nuclear magnetic resonance (NMR) spectroscopy, shedding light on the recognition mechanism of qRRMs with G-tract RNA. However, knowledge of the recognition details is still lacking. To investigate how qRRMs specifically bind with G-tract RNA and how the mutations of any guanine to an adenine in the G-tract affect the binding, molecular dynamics simulations with binding free energy analysis were performed based on the NMR structure of qRRM2 in complex with G-tract RNA. Simulation results demonstrate that qRRM2 binds strongly with G-tract RNA, but any mutation of the G-tract leads to a drastic reduction of the binding free energy. Further comparisons of the energetic components reveal that van der Waals and non-polar interactions play essential roles in the binding between qRRM2 and G-tract RNA, but the interactions are weakened by the effect of RNA mutations. Structural and dynamical analyses indicate that when qRRM2 binds with G-tract RNA, both qRRM2 and G-tract maintain stabilized structures and dynamics; however, the stability is disrupted by the mutations of the G-tract. These results provide novel insights into the recognition mechanism of qRRM2 with G-tract RNA that are not elucidated by the NMR technique. Copyright © 2017 Elsevier Inc. All rights reserved.

Hermes (Rbpms is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis

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Tristan Aguero

2016-01-01

Full Text Available The germ cell lineage in Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1, localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos-3′UTR. Importantly, Hermes/Rbpms specifically binds nanos, but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1. One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA.

Hermes (Rbpms) is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis.

Science.gov (United States)

Aguero, Tristan; Zhou, Yi; Kloc, Malgorzata; Chang, Patrick; Houliston, Evelyn; King, Mary Lou

2016-03-01

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1 , localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos -3'UTR. Importantly, Hermes/Rbpms specifically binds nanos , but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1 . One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA .

Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.

Science.gov (United States)

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

2005-01-07

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.

Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies

International Nuclear Information System (INIS)

Cauchi, Ruben J.; Sanchez-Pulido, Luis; Liu, Ji-Long

2010-01-01

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN, Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases we confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. We then made use of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Our cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport.

Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies

Energy Technology Data Exchange (ETDEWEB)

Cauchi, Ruben J.; Sanchez-Pulido, Luis [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX (United Kingdom); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX (United Kingdom)

2010-08-15

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN, Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases we confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. We then made use of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Our cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport.

Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

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Robinson Melvin L

2005-07-01

Full Text Available Abstract Background The Cajal body (CB is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs, which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. Results In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. Conclusion Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB.

The hnRNP-Q protein LIF2 participates in the plant immune response.

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Clémentine Le Roux

Full Text Available Eukaryotes have evolved complex defense pathways to combat invading pathogens. Here, we investigated the role of the Arabidopsis thaliana heterogeneous nuclear ribonucleoprotein (hnRNP-Q LIF2 in the plant innate immune response. We show that LIF2 loss-of-function in A. thaliana leads to changes in the basal expression of the salicylic acid (SA- and jasmonic acid (JA- dependent defense marker genes PR1 and PDF1.2, respectively. Whereas the expression of genes involved in SA and JA biosynthesis and signaling was also affected in the lif2-1 mutant, no change in SA and JA hormonal contents was detected. In addition, the composition of glucosinolates, a class of defense-related secondary metabolites, was altered in the lif2-1 mutant in the absence of pathogen challenge. The lif2-1 mutant exhibited reduced susceptibility to the hemi-biotrophic pathogen Pseudomonas syringae and the necrotrophic ascomycete Botrytis cinerea. Furthermore, the lif2-1 sid2-2 double mutant was less susceptible than the wild type to P. syringae infection, suggesting that the lif2 response to pathogens was independent of SA accumulation. Together, our data suggest that lif2-1 exhibits a basal primed defense state, resulting from complex deregulation of gene expression, which leads to increased resistance to pathogens with various infection strategies. Therefore, LIF2 may function as a suppressor of cell-autonomous immunity. Similar to its human homolog, NSAP1/SYNCRIP, a trans-acting factor involved in both cellular processes and the viral life cycle, LIF2 may regulate the conflicting aspects of development and defense programs, suggesting that a conserved evolutionary trade-off between growth and defense pathways exists in eukaryotes.

Predictions of RNA-binding ability and aggregation propensity of proteins

OpenAIRE

Agostini, Federico, 1985-

2014-01-01

RNA-binding proteins (RBPs) control the fate of a multitude of coding and non-coding transcripts. Formation of ribonucleoprotein (RNP) complexes fine-tunes regulation of post-transcriptional events and influences gene expression. Recently, it has been observed that non-canonical proteins with RNA-binding ability are enriched in structurally disordered and low-complexity regions that are generally involved in functional and dysfunctional associations. Therefore, it is possible that interaction...

Engineering of blood vessel patterns by angio-morphogens [angiotropins]: non-mitogenic copper-ribonucleoprotein cytokins [CuRNP ribokines] with their metalloregulated constituents of RAGE-binding S100-EF-hand proteins and extracellular RNA bioaptamers in vascular remodeling of tissue and angiogenesis in vitro

Energy Technology Data Exchange (ETDEWEB)

Wissler, J.H. [ARCONS Applied Research, Bad Nauheim (Germany)

2001-12-01

Tissue vascularization is requisite to successful cell-based therapies, biomaterial design and implant integration. Thus, known problems in ossointegration of avascular implants in connection with the generation of bone tissue reflect arrays of general problems of socio-economic relevance existing in reparative medicine still waiting for to be solved. For this purpose, morphogenesis and remodeling of endothelial angio-architectures in tissue and in vitro by isolated non-mitogenic angio-morphogens [angiotropins] are considered in terms of their structure, function and action mechanisms. Extracellular angiotropins are secreted by activated leukocytes/monocytes/macrophages. They are a family of cytokines with morphogen bioactivity selectively directed to endothelial cells. Their structure was deciphered as metalloregulated copper-ribonucleoproteins [CuRNP ribokines]. They are built up of angiotropin-related S100-EF-hand protein [ARP] and highly modified and edited 5'end-phosphorylated RNA [ARNA], complexed together by copper ions. Oxidant-sensitive ARNA and their precursors represent novel types in a RNA world: They are the first isolated and sequenced forms of extracellular RNA [eRNA], may act as cytokine and bioaptamer, contain isoguanosine [crotonoside] as modified nucleoside and show up copper as RNA-structuring transition metal ion. By metalloregulated bioaptamer functions, ARNA impart novel biofunctions to RAGE-binding S100-EF-hand proteins. Angiotropin morphogens were shown suitable for neointiation and remodeling of blood vessel patterns in different, adult, embryonal and artificial tissues. These neovascular patterns manifest regulated hemodynamics for preventing tissue necrosis, supporting tissue functions and promoting wound healing. As evaluated in skin and muscle vascularization, the neovascular patterns are integrated into homeostatic control mechanisms of tissue. Thus, the morphogens show up beneficial perspectives and are suggested useful tools

Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

Science.gov (United States)

Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

2013-01-01

Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

Cooperative RNP assembly: Complementary rescue of structural defects by protein and RNA subunits of archaeal RNase P

Science.gov (United States)

Chen, Wen-Yi; Xu, Yiren; Cho, I-Ming; Oruganti, Sri Vidya; Foster, Mark P.; Gopalan, Venkat

2011-01-01

RNase P is a ribonucleoprotein (RNP) complex that utilizes a Mg2+-dependent RNA catalyst to cleave the 5′-leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg2+ coordination, and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5•RPP30 and RPP21•RPP29). In this study, we employed a previously characterized substrate-enzyme conjugate [pre-tRNATyr-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNATyr-MjaΔU RPR compared to the wildtype, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P towards its functional conformation. (236 words) PMID:21683084

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

Science.gov (United States)

Goncharov, I; Palfi, Z; Bindereif, A; Michaeli, S

1999-04-30

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

VHL genetic alteration in CCRCC does not determine de-regulation of HIF, CAIX, hnRNP A2/B1 and osteopontin.

LENUS (Irish Health Repository)

Nyhan, Michelle J

2012-01-31

BACKGROUND: von Hippel-Lindau (VHL) tumour suppressor gene inactivation is associated with clear cell renal cell carcinoma (CCRCC) development. The VHL protein (pVHL) has been proposed to regulate the expression of several proteins including Hypoxia Inducible Factor-alpha (HIF-alpha), carbonic anhydrase (CA)IX, heterogeneous nuclear ribonucleoprotein (hnRNP) A2\\/B1 and osteopontin. pVHL has been characterized in vitro, however, clinical studies are limited. We evaluated the impact of VHL genetic alterations on the expression of several pVHL protein targets in paired normal and tumor tissue. METHODS: The VHL gene was sequenced in 23 CCRCC patients and VHL transcript levels were evaluated by real-time RT-PCR. Expression of pVHL\\'s protein targets were determined by Western blotting in 17 paired patient samples. RESULTS: VHL genetic alterations were identified in 43.5% (10\\/23) of CCRCCs. HIF-1alpha, HIF-2alpha and CAIX were up-regulated in 88.2% (15\\/17), 100% (17\\/17) and 88.2% (15\\/17) of tumors respectively and their expression is independent of VHL status. hnRNP A2\\/B1 and osteopontin expression was variable in CCRCCs and had no association with VHL genetic status. CONCLUSION: As expression of these proposed pVHL targets can be achieved independently of VHL mutation (and possibly by hypoxia alone), these data suggests that other pVHL targets may be more crucial in renal carcinogenesis.

B-CELL EPITOPE ON THE U1 SNRNP-C AUTOANTIGEN CONTAINS A SEQUENCE SIMILAR TO THAT OF THE HERPES-SIMPLEX VIRUS PROTEIN

NARCIS (Netherlands)

MISAKI, Y; YAMAMOTO, K; YANAGI, K; MIURA, H; ICHIJO, H; KATO, T; MATO, T; WELLINGWESTER, S; NISHIOKA, K; ITO, K

The mechanism of autoantibody production in autoimmune diseases is not well understood. In the present study we performed the B cell epitope mapping of the U1 small nuclear ribonucleoprotein (snRNP)-C, one of the target molecules of anti-nRNP autoantibody to investigate how B cells respond to the

Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering

International Nuclear Information System (INIS)

Sickmier, E. Allen; Frato, Katherine E.; Kielkopf, Clara L.

2006-01-01

A complex of the essential splicing factor U2AF 65 and a deoxyuridine oligonucleotide has been crystallized by modification of an interdomain linker. The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF 65 ) binds the polypyrimidine tract near the 3′ splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF 65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein–nucleic acid complex. Cocrystals of a U2AF 65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 Å resolution and contain one complex per asymmetric unit

Substrate recognition by ribonucleoprotein ribonuclease MRP.

Science.gov (United States)

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

2011-02-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

International Nuclear Information System (INIS)

Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

2011-01-01

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development

Human T-lymphotropic virus type 1 Tax protein complexes with P-TEFb and competes for Brd4 and 7SK snRNP/HEXIM1 binding.

Science.gov (United States)

Cho, Won-Kyung; Jang, Moon Kyoo; Huang, Keven; Pise-Masison, Cynthia A; Brady, John N

2010-12-01

Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both in vitro using purified Tax protein and in vivo in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.

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

Science.gov (United States)

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

2013-07-01

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

RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection

International Nuclear Information System (INIS)

Kleinschmidt, A.M.; Pederson, T.

1990-01-01

The authors present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32 P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells

Production of Purified CasRNPs for Efficacious Genome Editing.

Science.gov (United States)

Lingeman, Emily; Jeans, Chris; Corn, Jacob E

2017-10-02

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

Science.gov (United States)

Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

2014-11-01

In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

The TROVE module: a common element in Telomerase, Ro and Vault ribonucleoproteins.

Science.gov (United States)

Bateman, Alex; Kickhoefer, Valerie

2003-10-16

Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding. The TROVE module--Telomerase, Ro and Vault module--is found in TEP1 and Ro60 the protein components of three ribonucleoprotein particles. This novel module, consisting of one or more domains, may be involved in binding the RNA components of the three RNPs, which are telomerase RNA, Y RNA and vault RNA. A second conserved region in these proteins is shown to be a member of the vWA domain family. The vWA domain in TEP1 is closely related to the previously recognised vWA domain in VPARP a second component of the vault particle. This vWA domain may mediate interactions between these vault components or bind as yet unidentified components of the RNPs. This work suggests that a number of ribonucleoprotein components use a common RNA-binding module. The TROVE module is also found in bacterial ribonucleoproteins suggesting an ancient origin for these ribonucleoproteins.

RNA and Proteins: Mutual Respect [version 1; referees: 3 approved

Directory of Open Access Journals (Sweden)

Kathleen B. Hall

2017-03-01

Full Text Available Proteins and RNA are often found in ribonucleoprotein particles (RNPs, where they function in cellular processes to synthesize proteins (the ribosome, chemically modify RNAs (small nucleolar RNPs, splice pre-mRNAs (the spliceosome, and, on a larger scale, sequester RNAs, degrade them, or process them (P bodies, Cajal bodies, and nucleoli. Each RNA–protein interaction is a story in itself, as both molecules can change conformation, compete for binding sites, and regulate cellular functions. Recent studies of Xist long non-coding RNP, the U4/5/6 tri-small nuclear RNP complex, and an activated state of a spliceosome reveal new features of RNA interactions with proteins, and, although their stories are incomplete, they are already fascinating.

Amino acid substitutions affecting aspartic acid 605 and valine 606 decrease the interaction strength between the influenza virus RNA polymerase PB2 '627' domain and the viral nucleoprotein.

Science.gov (United States)

Hsia, Ho-Pan; Yang, Yin-Hua; Szeto, Wun-Chung; Nilsson, Benjamin E; Lo, Chun-Yeung; Ng, Andy Ka-Leung; Fodor, Ervin; Shaw, Pang-Chui

2018-01-01

The influenza virus RNA genome is transcribed and replicated in the context of the viral ribonucleoprotein (vRNP) complex by the viral RNA polymerase. The nucleoprotein (NP) is the structural component of the vRNP providing a scaffold for the viral RNA. In the vRNP as well as during transcription and replication the viral polymerase interacts with NP but it is unclear which parts of the polymerase and NP mediate these interactions. Previously the C-terminal '627' domain (amino acids 538-693) of PB2 was shown to interact with NP. Here we report that a fragment encompassing amino acids 146-185 of NP is sufficient to mediate this interaction. Using NMR chemical shift perturbation assays we show that amino acid region 601 to 607 of the PB2 '627' domain interacts with this fragment of NP. Substitutions of these PB2 amino acids resulted in diminished RNP activity and surface plasmon resonance assays showed that amino acids D605 was essential for the interaction with NP and V606 may also play a partial role in the interaction. Collectively these results reveal a possible interaction surface between NP and the PB2 subunit of the RNA polymerase complex.

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft

Directory of Open Access Journals (Sweden)

Naoki Takizawa

2016-09-01

Full Text Available The influenza glycoproteins, hemagglutinin (HA and neuraminidase (NA, which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1 in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft.

Science.gov (United States)

Takizawa, Naoki; Momose, Fumitaka; Morikawa, Yuko; Nomoto, Akio

2016-09-10

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5′ End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

Science.gov (United States)

Wang, Dai; Parrish, Colin R.

1999-01-01

Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5′ end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5′ end viral genome did not affect replication of the virus. PMID:10438866

N-methylation of the heterogeneous nuclear ribonucleoproteins in HeLa cells

International Nuclear Information System (INIS)

Rieker, J.P.

1984-01-01

Several of the core proteins on the 40S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from HeLa cells contain N/sup G/,N/sup G/-dimethyl-L-arginine (uDMA). 3-deazaadenosine (c 3 Ado), an inhibitor of and substrate for s-adenosyl-L-homocysteine hydrolase, has been used to study the methylation patterns of the individual polypeptides. Trimethyllysine and uDMA formation in total cellular protein were inhibited in the presence of the drug while other methylated basic amino acids were unaffected. This inhibition was reversed within 60 min after removal of the drug from the medium. Monolayer HeLa cultures were incubated with [methyl- 3 H]-L-methoinine for 12 hours in the presence of 50 uM c 3 Ado. Purified particles were obtained by centrifugation of nuclear extracts on sucrose density gradients. The core proteins were isolated by two-dimensional gel electrophoresis, acid hydrolyzed and analyzed for radioactivity incorporated into methionine and methylated basic amino acids. The ratio of radioactivity incorporated into uDMA relative to that into methionine for the two major particle proteins with molecular weights of 31,000 (A 1 ) and 43,000 (A 2 ) was about 2.0 and 0.2 in control cultures. In the presence of c 3 Ado, these ratios were depressed 60 to 80%. Results of pulse-chase experiments suggested that A 1 and A 2 are metabolically stable proteins (t/sub 0.5/ > 75 hr), whether or not the proteins were undermethylated. Monomethyl-L-arginine may be a precursor in the formation of u-DMA

The TROVE module: A common element in Telomerase, Ro and Vault ribonucleoproteins

Directory of Open Access Journals (Sweden)

Bateman Alex

2003-10-01

Full Text Available Abstract Background Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding. Results The TROVE module – Telomerase, Ro and Vault module – is found in TEP1 and Ro60 the protein components of three ribonucleoprotein particles. This novel module, consisting of one or more domains, may be involved in binding the RNA components of the three RNPs, which are telomerase RNA, Y RNA and vault RNA. A second conserved region in these proteins is shown to be a member of the vWA domain family. The vWA domain in TEP1 is closely related to the previously recognised vWA domain in VPARP a second component of the vault particle. This vWA domain may mediate interactions between these vault components or bind as yet unidentified components of the RNPs. Conclusions This work suggests that a number of ribonucleoprotein components use a common RNA-binding module. The TROVE module is also found in bacterial ribonucleoproteins suggesting an ancient origin for these ribonucleoproteins.

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

Directory of Open Access Journals (Sweden)

C. Soldani

2010-05-01

Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

Integration of mRNP formation and export.

Science.gov (United States)

Björk, Petra; Wieslander, Lars

2017-08-01

Expression of protein-coding genes in eukaryotes relies on the coordinated action of many sophisticated molecular machineries. Transcription produces precursor mRNAs (pre-mRNAs) and the active gene provides an environment in which the pre-mRNAs are processed, folded, and assembled into RNA-protein (RNP) complexes. The dynamic pre-mRNPs incorporate the growing transcript, proteins, and the processing machineries, as well as the specific protein marks left after processing that are essential for export and the cytoplasmic fate of the mRNPs. After release from the gene, the mRNPs move by diffusion within the interchromatin compartment, making up pools of mRNPs. Here, splicing and polyadenylation can be completed and the mRNPs recruit the major export receptor NXF1. Export competent mRNPs interact with the nuclear pore complex, leading to export, concomitant with compositional and conformational changes of the mRNPs. We summarize the integrated nuclear processes involved in the formation and export of mRNPs.

Radioimmunoassay for antibodies to rubella virus and its ribonucleoprotein component

International Nuclear Information System (INIS)

Ho-Terry, L.; Cohen, A.

1979-01-01

Using a radioimmune precipitation technique, the antibody response to intact rubella virus and its ribonucleoprotein component was measured. The method was very sensitive and reproducible, and did not require preliminary serum fractionation for the identification of antibodies of different immunoglobulin classes. The results showed that the IgA and IgG antibodies against the intact virus persisted in the sera of patients long after the initial infection. In contrast, IgA and IgG antibodies against the ribonucleoprotein component of rubella virus were detected only in sera of patients after recent rubella infection. This observation suggested that a test for antibodies to the ribonucleoprotein component may provide additional evidence in the diagnosis of recent rubella infection. This could be potentially a useful test particularly in the management of pregnant patients. (U.K.)

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes

DEFF Research Database (Denmark)

Honoré, B; Rasmussen, H H; Vorum, H

1995-01-01

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share...... epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75......%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used...

Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

DEFF Research Database (Denmark)

Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas

2016-01-01

for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an i...... downstream of the 5' splice site can be blocked by SSOs to activate the exon. CONCLUSIONS: The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease...

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

Science.gov (United States)

Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

2018-04-01

Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Chaperoning 5S RNA assembly.

Science.gov (United States)

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

2015-07-01

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

Formation of virions is strictly required for turnip yellows virus long-distance movement in plants.

Science.gov (United States)

Hipper, Clémence; Monsion, Baptiste; Bortolamiol-Bécet, Diane; Ziegler-Graff, Véronique; Brault, Véronique

2014-02-01

Viral genomic RNA of the Turnip yellows virus (TuYV; genus Polerovirus; family Luteoviridae) is protected in virions formed by the major capsid protein (CP) and the minor component, the readthrough (RT*) protein. Long-distance transport, used commonly by viruses to systemically infect host plants, occurs in phloem sieve elements and two viral forms of transport have been described: virions and ribonucleoprotein (RNP) complexes. With regard to poleroviruses, virions have always been presumed to be the long-distance transport form, but the potential role of RNP complexes has not been investigated. Here, we examined the requirement of virions for polerovirus systemic movement by analysing CP-targeted mutants that were unable to form viral particles. We confirmed that TuYV mutants that cannot encapsidate into virions are not able to reach systemic leaves. To completely discard the possibility that the introduced mutations in CP simply blocked the formation or the movement of RNP complexes, we tested in trans complementation of TuYV CP mutants by providing WT CP expressed in transgenic plants. WT CP was able to facilitate systemic movement of TuYV CP mutants and this observation was always correlated with the formation of virions. This demonstrated clearly that virus particles are essential for polerovirus systemic movement.

Cup regulates oskar mRNA stability during oogenesis.

Science.gov (United States)

Broyer, Risa M; Monfort, Elena; Wilhelm, James E

2017-01-01

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

Genome-wide analysis of uncapped mRNAs under heat stress in Arabidopsis

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Emilio Gutierrez-Beltran

2015-09-01

Full Text Available Recently, we have showed that Tudor Staphylococcal Nuclease (TSN or Tudor-SN proteins (TSN1 and TSN2 are localized in cytoplasmic messenger ribonucleoprotein (mRNP complexes called stress granules (SG and processing bodies (PB under heat stress in Arabidopsis. One of the primary functions of these mRNP complexes is mRNA decay, which generates uncapped mRNAs by the action of endonucleases and decapping enzymes (Thomas et al., 2011 [1]. In order to figure out whether TSN proteins could be implicated in mRNA decay, we isolated uncapped and total mRNAs of Wild type (WT; Col and Ler and TSN double knock-out (tsn1tsn2 seedlings grown under heat stress (39 °C for 40 min and control (23 °C conditions. Here, we provide the experimental procedure to reproduce the results (NCBI GEO accession number GSE63522 published by Gutierrez-Beltran et al. (2015 in The Plant Cell [2].

Vanillin inhibits translation and induces messenger ribonucleoprotein (mRNP) granule formation in saccharomyces cerevisiae: application and validation of high-content, image-based profiling.

Science.gov (United States)

Iwaki, Aya; Ohnuki, Shinsuke; Suga, Yohei; Izawa, Shingo; Ohya, Yoshikazu

2013-01-01

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.

Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL.

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Cui, Yunxi; Koirala, Deepak; Kang, HyunJin; Dhakal, Soma; Yangyuoru, Philip; Hurley, Laurence H; Mao, Hanbin

2014-05-01

Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120-180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U

DEFF Research Database (Denmark)

Davis, Matti; Hatzubai, Ada; Andersen, Jens S

2002-01-01

in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished....... Consequently, hnRNP-U engages a highly neddylated active SCF(beta-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold...

The Cajal body and the nucleolus: “In a relationship” or “It's complicated”?

Science.gov (United States)

2017-01-01

ABSTRACT From their initial identification as ‘nucleolar accessory bodies’ more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the 2 structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed. PMID:27661468

The Cajal body and the nucleolus: "In a relationship" or "It's complicated"?

Science.gov (United States)

Trinkle-Mulcahy, Laura; Sleeman, Judith E

2017-06-03

From their initial identification as 'nucleolar accessory bodies' more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the 2 structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed.

EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin.

Science.gov (United States)

Mohan Kumar, Dipu; Lin, Mingqun; Xiong, Qingming; Webber, Mathew James; Kural, Comert; Rikihisa, Yasuko

2015-11-03

Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization. Ehrlichia chaffeensis, an

The recruitment of the U5 snRNP to nascent transcripts requires internal loop 1 of U5 snRNA.

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Kim, Rebecca; Paschedag, Joshua; Novikova, Natalya; Bellini, Michel

2012-12-01

In this study, we take advantage of the high spatial resolution offered by the nucleus and lampbrush chromosomes of the amphibian oocyte to investigate the mechanisms that regulate the intranuclear trafficking of the U5 snRNP and its recruitment to nascent transcripts. We monitor the fate of newly assembled fluorescent U5 snRNP in Xenopus oocytes depleted of U4 and/or U6 snRNAs and demonstrate that the U4/U6.U5 tri-snRNP is not required for the association of U5 snRNP with Cajal bodies, splicing speckles, and nascent transcripts. In addition, using a mutational analysis, we show that a non-functional U5 snRNP can associate with nascent transcripts, and we further characterize internal loop structure 1 of U5 snRNA as a critical element for licensing U5 snRNP to target both nascent transcripts and splicing speckles. Collectively, our data support the model where the recruitment of snRNPs onto pre-mRNAs is independent of spliceosome assembly and suggest that U5 snRNP may promote the association of the U4/U6.U5 tri-snRNP with nascent transcripts.

Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

Science.gov (United States)

Abdeldaim, Guma; Svensson, Erik; Blomberg, Jonas; Herrmann, Björn

2016-11-01

A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

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Helena Hernández

2009-09-01

Full Text Available Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts.

Science.gov (United States)

Lisse, Thomas S; Vadivel, Kanagasabai; Bajaj, S Paul; Chun, Rene F; Hewison, Martin; Adams, John S

2014-01-01

Heterogeneous nuclear ribonucleoprotein (hnRNP) C plays a key role in RNA processing. More recently hnRNP C has also been shown to function as a DNA binding protein exerting a dominant-negative effect on transcriptional responses to the vitamin D hormone,1,25-dihydroxyvitamin D (1,25(OH) 2 D), via interaction in cis with vitamin D response elements (VDREs). The physiologically active form of human hnRNPC is a tetramer of hnRNPC1 (huC1) and C2 (huC2) subunits known to be critical for specific RNA binding activity in vivo , yet the requirement for heterodimerization of huC1 and C2 in DNA binding and downstream action is not well understood. While over-expression of either huC1 or huC2 alone in mouse osteoblastic cells did not suppress 1,25(OH) 2 D-induced transcription, over-expression of huC1 and huC2 in combination using a bone-specific polycistronic vector successfully suppressed 1,25(OH) 2 D-mediated induction of osteoblast target gene expression. Over-expression of either huC1 or huC2 in human osteoblasts was sufficient to confer suppression of 1,25(OH) 2 D-mediated transcription, indicating the ability of transfected huC1 and huC2 to successfully engage as heterodimerization partners with endogenously expressed huC1 and huC2. The failure of the chimeric combination of mouse and human hnRNPCs to impair 1,25(OH) 2 D-driven gene expression in mouse cells was structurally predicted, owing to the absence of the last helix in the leucine zipper (LZ) heterodimerization domain of hnRNPC gene product in lower species, including the mouse. These results confirm that species-specific heterodimerization of hnRNPC1 and hnRNPC2 is a necessary prerequisite for DNA binding and down-regulation of 1,25(OH) 2 D-VDR-VDRE-directed gene transactivation in osteoblasts.

U3 snoRNP associates with fibrillarin a component of the scleroderma clumpy nucleolar domain

DEFF Research Database (Denmark)

Herrera-Esparza, Rafael; Kruse, Lars; von Essen, Marina

2002-01-01

by ELISA was recognized by the clumpy scleroderma serum from the majority of patients. In situ hybridization assays showed that the fibrillarin tagged by the elicited antibodies was colocalized with U3 snoRNP in the nucleolus in a clumpy manner and coprecipitated the U3 snoRNP. In conclusion...

Germ Plasm Anchoring Is a Dynamic State that Requires Persistent Trafficking

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Kristina S. Sinsimer

2013-12-01

Full Text Available Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, we show that RNP particle motility is required for long-term germ plasm retention. We propose that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations.

The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate Cancer Treatment Assignment

Science.gov (United States)

2017-10-01

for prostate cancer , the work may impact development of diagnostic /prognostic products based on pThoc1. The presence of the THO ribonucleoprotin...AWARD NUMBER: W81XWH-14-1-0475 TITLE: The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate Cancer Treatment Assignment PRINCIPAL...15Sept 2016 - 14Sep2017 4. TITLE AND SUBTITLE The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate 5a. CONTRACT NUMBER Cancer Treatment

Procedure za prilaz i sletanje na aerodrome 'Nikola Tesla' i 'Batajnica' primenom RNP AR APCH i Baro-VNAV / Approach and landing procedures for airports 'Nikola Tesla' and 'Batajnica' using RNP AR APCH and Baro-VNAV

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Vojislav S. Milosavljević

2010-01-01

a conventional approach. The airports Both of the airports have their own TMA, but currently there are conventional SID and STAR flows for 'Nikola Tesla' running through TMA 'Batajnica' while procedures for 'Batajnica' are issued only as temporary for special flights. Procedure design A detailed overview of procedure design includes general considerations, the principles on which the design is based, design of turns, maps of inbound flows featuring holdings and missed approach segments. Holdings All holdings, including both in approach and in missed approach for both airports are defined in accordance with RNP holding criteria and in a way that aircraft inbound from as many as possible flows could use them. Final approaches All final approaches consist of a single TF leg (Track to Fix. Containment areas are linear and all their elements are determined by RNP 0.3. Baro-VNAV is used for vertical guidance. Missed approach segments with MAP holdings In design of missed approach segment only TF legs were used for a straight flight and RF legs for turns. The priority was minimization of total complexity in the segment which is why the minimal number of legs was used and turn amplitude was limited. Conclusion Implementation of RNP AR APCH in combination with Baro- VNAV and CDA at 'Nikola Tesla' and 'Batajnica' would bring many benefits without any observed negative side effects. Note The topic is addressed with far more details in diploma thesis of Vojislav Milosavljević, originally titled 'Procedure za prilaz i sletanje na aerodrome 'Nikola Tesla' i 'Batajnica' primenom RNP AR APCH i Baro-VNAV' defended at The Faculty of Traffic and Transport Engineering, Belgrade, Serbia, on July, 7th 2008. Detailed calculations, figures, explanations of chosen solutions and other are featured in the thesis available at the library of the faculty.

Functional organization of the Sm core in the crystal structure of human U1 snRNP.

OpenAIRE

Weber, G.; Trowitzsch, S.; Kastner, B.; Lührmann, R.; Wahl, M.

2010-01-01

The U1 small nuclear ribonucleoprotein initiates the assembly of the spliceosome. Here, the structure of the natively purified U1 small nuclear ribonucleoprotein particle reveals the core Sm protein ring and its interactions with the Sm site in the small nuclear RNA.

Dynein-dependent transport of nanos RNA in Drosophila sensory neurons requires Rumpelstiltskin and the germ plasm organizer Oskar.

Science.gov (United States)

Xu, Xin; Brechbiel, Jillian L; Gavis, Elizabeth R

2013-09-11

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-type nos mRNA but not by a localization-compromised nos derivative. While localization is essential for nos function in dendrite morphogenesis, the mechanism underlying the transport of nos RNP particles was unknown. We investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labeled nos mRNA. We show that dynein motor machinery components mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization of nos during oogenesis, also function in da neurons for formation and transport of nos RNP particles. Additionally, we show that nos regulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts.

Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity

International Nuclear Information System (INIS)

Zhirnov, O.P.; Manykin, A.A.; Rossman, J.S.; Klenk, H.D.

2016-01-01

Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivity whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell. - Highlights: • The influenza A virus has a novel asymmetric internal structure. • The structure is largely maintained by M1-RNP cohesion within the virion. • This asymmetry plays an important role during viral entry, facilitating virus uncoating and the initiation of a productive

Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity

Energy Technology Data Exchange (ETDEWEB)

Zhirnov, O.P., E-mail: zhirnov@inbox.ru [D.I. Ivanovsky Institute of Virology, Moscow 123098 (Russian Federation); Manykin, A.A. [D.I. Ivanovsky Institute of Virology, Moscow 123098 (Russian Federation); Rossman, J.S. [School of Biosciences, University of Kent, Canterbury CT27NJ (United Kingdom); Klenk, H.D. [Institute of Virology, Philipps University, Marburg 35037 (Germany)

2016-05-15

Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivity whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell. - Highlights: • The influenza A virus has a novel asymmetric internal structure. • The structure is largely maintained by M1-RNP cohesion within the virion. • This asymmetry plays an important role during viral entry, facilitating virus uncoating and the initiation of a productive

Biomass conversion inhibitors furfural and 5-hydroxymethylfurfural induce formation of messenger RNP granules and attenuate translation activity in Saccharomyces cerevisiae.

Science.gov (United States)

Iwaki, Aya; Kawai, Takao; Yamamoto, Yosuke; Izawa, Shingo

2013-03-01

Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.

Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients [v2; ref status: indexed, http://f1000r.es/4dh

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Sangmin Lee

2014-09-01

Full Text Available Some somatic single nucleotide variants (SNVs are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1, an autoantigen associated with multiple sclerosis (MS would contain SNVs. MS patients develop antibodies to hnRNP A1293-304, an epitope within the M9 domain (AA268-305 of hnRNP A1. M9 is hnRNP A1’s nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1 and allows for hnRNP A1’s transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1268-289 and the MS IgG epitope (hnRNP A1293-304, within M9.  In contrast to the nuclear localization of wild type (WT hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.

Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients [v1; ref status: indexed, http://f1000r.es/3nv

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Sangmin Lee

2014-06-01

Full Text Available Some somatic single nucleotide variants (SNVs are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1, an autoantigen associated with multiple sclerosis (MS would contain SNVs. MS patients develop antibodies to hnRNP A1293-304, an epitope within the M9 domain (AA268-305 of hnRNP A1. M9 is hnRNP A1’s nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1 and allows for hnRNP A1’s transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1268-289 and the MS IgG epitope (hnRNP A1293-304, within M9.  In contrast to the nuclear localization of wild type (WT hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.

Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast

DEFF Research Database (Denmark)

Nielsen, Klaus H; Valásek, Leos; Sykes, Caroah

2006-01-01

We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation...

60 kD Ro and nRNP A frequently initiate human lupus autoimmunity.

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Latisha D Heinlen

2010-03-01

Full Text Available Systemic lupus erythematosus (SLE is a clinically heterogeneous, humoral autoimmune disorder. The unifying feature among SLE patients is the production of large quantities of autoantibodies. Serum samples from 129 patients collected before the onset of SLE and while in the United States military were evaluated for early pre-clinical serologic events. The first available positive serum sample frequently already contained multiple autoantibody specificities (65%. However, in 34 SLE patients the earliest pre-clinical serum sample positive for any detectable common autoantibody bound only a single autoantigen, most commonly 60 kD Ro (29%, nRNP A (24%, anti-phospholipids (18% or rheumatoid factor (15%. We identified several recurrent patterns of autoantibody onset using these pre-diagnostic samples. In the serum samples available, anti-nRNP A appeared before or simultaneously with anti-nRNP 70 K in 96% of the patients who had both autoantibodies at diagnosis. Anti-60 kD Ro antibodies appeared before or simultaneously with anti-La (98% or anti-52 kD Ro (95%. The autoantibody response in SLE patients begins simply, often binding a single specific autoantigen years before disease onset, followed by epitope spreading to additional autoantigenic specificities that are accrued in recurring patterns.

Hypothesis: A Role for Fragile X Mental Retardation Protein in Mediating and Relieving MicroRNA-Guided Translational Repression?

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Isabelle Plante

2006-01-01

Full Text Available MicroRNA (miRNA-guided messenger RNA (mRNA translational repression is believed to be mediated by effector miRNA-containing ribonucleoprotein (miRNP complexes harboring fragile X mental retardation protein (FMRP. Recent studies documented the nucleic acid chaperone properties of FMRP and characterized its role and importance in RNA silencing in mammalian cells. We propose a model in which FMRP could facilitate miRNA assembly on target mRNAs in a process involving recognition of G quartet structures. Functioning within a duplex miRNP, FMRP may also mediate mRNA targeting through a strand exchange mechanism, in which the miRNA* of the duplex is swapped for the mRNA. Furthermore, FMRP may contribute to the relief of miRNA-guided mRNA repression through a reverse strand exchange reaction, possibly initiated by a specific cellular signal, that would liberate the mRNA for translation. Suboptimal utilization of miRNAs may thus account for some of themolecular defects in patients with the fragile X syndrome.

The exosome associates cotranscriptionally with the nascent pre-mRNP through interactions with heterogeneous nuclear ribonucleoproteins

DEFF Research Database (Denmark)

Hessle, Viktoria; Björk, Petra; Sokolowski, Marcus

2009-01-01

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality check...

Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function

International Nuclear Information System (INIS)

Asai, Kengo; Platt, Craig; Cochrane, Alan

2003-01-01

The control of HIV-1 viral RNA splicing and transport plays an important role in the successful replication of the virus. Previous studies have identified both an exon splicing enhancer (ESE) and a bipartite exon splicing silencer (ESS3a and ESS3b) within the terminal exon of HIV-1 that are involved in modulating both splicing and Rev-mediated export of viral RNA. To define the mechanism of ESS3a function, experiments were carried out to better define the cis and trans components required for ESS3a activity. Mutations throughout the 30-nt element resulted in partial loss of ESS function. Combining mutations was found to have an additive effect, suggesting the presence of multiple binding sites. Analysis of interacting factors identified hnRNP A1 as one component of the complex that modulates ESS3a activity. However, subsequent binding analyses determined that hnRNP A1 interacts with only one portion of ESS3a, suggesting the involvement of another host factor. Parallel analysis of the effect of the mutations on Rev-mediated export determined that there is not a direct correlation between the effect of the mutations on splicing and RNA transport. Consistent with this hypothesis, replacement of ESS3a with consensus hnRNP A1 binding sites was found to be insufficient to block Rev-mediated RNA export

Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.

Science.gov (United States)

Berman, Andrea J; Gooding, Anne R; Cech, Thomas R

2010-10-01

The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Taken together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.

Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

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Jarrous, Nayef; Wolenski, Joseph S.; Wesolowski, Donna; Lee, Christopher; Altman, Sidney

1999-01-01

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. PMID:10444065

Connections between transcription, mRNP assembly and quality control in S. cerevisiae

DEFF Research Database (Denmark)

Jensen, Torben Heick

in the context of THO and rna14-3 mutants improves mRNP quality by acting upstream of transcription-site retention and nuclear degradation of the transcripts. As Rad3p mutant effects can be phenocopied by other mutations known to affect transcription and by the addition of transcription elongation drugs, our...

Influenza NA and PB1 Gene Segments Interact during the Formation of Viral Progeny: Localization of the Binding Region within the PB1 Gene

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Brad Gilbertson

2016-08-01

Full Text Available The influenza A virus genome comprises eight negative-sense viral RNAs (vRNAs that form individual ribonucleoprotein (RNP complexes. In order to incorporate a complete set of each of these vRNAs, the virus uses a selective packaging mechanism that facilitates co-packaging of specific gene segments but whose molecular basis is still not fully understood. Recently, we used a competitive transfection model where plasmids encoding the A/Puerto Rico/8/34 (PR8 and A/Udorn/307/72 (Udorn PB1 gene segments were competed to show that the Udorn PB1 gene segment is preferentially co-packaged into progeny virions with the Udorn NA gene segment. Here we created chimeric PB1 genes combining both Udorn and PR8 PB1 sequences to further define the location within the Udorn PB1 gene that drives co-segregation of these genes and show that nucleotides 1776–2070 of the PB1 gene are crucial for preferential selection. In vitro assays examining specific interactions between Udorn NA vRNA and purified vRNAs transcribed from chimeric PB1 genes also supported the importance of this region in the PB1-NA interaction. Hence, this work identifies an association between viral genes that are co-selected during packaging. It also reveals a region potentially important in the RNP-RNP interactions within the supramolecular complex that is predicted to form prior to budding to allow one of each segment to be packaged in the viral progeny. Our study lays the foundation to understand the co-selection of specific genes, which may be critical to the emergence of new viruses with pandemic potential.

Functional Interaction Map of Lyssavirus Phosphoprotein: Identification of the Minimal Transcription Domains

Science.gov (United States)

Jacob, Yves; Real, Eléonore; Tordo, Noël

2001-01-01

Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein–reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template. PMID:11559793

The crystal structure and RNA-binding of an orthomyxovirus nucleoprotein.

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Wenjie Zheng

2013-09-01

Full Text Available Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV (genus Isavirus. As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.

A Function for the hnRNP A1/A2 Proteins in Transcription Elongation.

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Lemieux, Bruno; Blanchette, Marco; Monette, Anne; Mouland, Andrew J; Wellinger, Raymund J; Chabot, Benoit

2015-01-01

The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.

A Function for the hnRNP A1/A2 Proteins in Transcription Elongation.

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Bruno Lemieux

Full Text Available The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.

Identification of species of viridans group streptococci in clinical blood culture isolates by sequence analysis of the RNase P RNA gene, rnpB.

Science.gov (United States)

Westling, Katarina; Julander, Inger; Ljungman, Per; Vondracek, Martin; Wretlind, Bengt; Jalal, Shah

2008-03-01

Viridans group streptococci (VGS) cause severe diseases such as infective endocarditis and septicaemia. Genetically, VGS species are very close to each other and it is difficult to identify them to species level with conventional methods. The aims of the present study were to use sequence analysis of the RNase P RNA gene (rnpB) to identify VGS species in clinical blood culture isolates, and to compare the results with the API 20 Strep system that is based on phenotypical characteristics. Strains from patients with septicaemia or endocarditis were analysed with PCR amplification and sequence analysis of the rnpB gene. Clinical data were registered as well. One hundred and thirty two VGS clinical blood culture isolates from patients with septicaemia (n=95) or infective endocarditis (n=36) were analysed; all but one were identified by rnpB. Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii strains were most common in the patients with infective endocarditis. In the isolates from patients with haematological diseases, Streptococcus mitis and S. oralis dominated. In addition in 76 of the isolates it was possible to compare the results from rnpB analysis and the API 20 Strep system. In 39/76 (51%) of the isolates the results were concordant to species level; in 55 isolates there were no results from API 20 Strep. Sequence analysis of the RNase P RNA gene (rnpB) showed that almost all isolates could be identified. This could be of importance for evaluation of the portal of entry in patients with septicaemia or infective endocarditis.

Three-dimensional structure of a pre-catalytic human spliceosomal complex B.

Science.gov (United States)

Boehringer, Daniel; Makarov, Evgeny M; Sander, Bjoern; Makarova, Olga V; Kastner, Berthold; Lührmann, Reinhard; Stark, Holger

2004-05-01

Major structural changes occur in the spliceosome during its transition from the fully assembled complex B to the catalytically activated spliceosome. To understand the rearrangement, it is necessary to know the detailed three-dimensional structures of these complexes. Here, we have immunoaffinity-purified human spliceosomes (designated B Delta U1) at a stage after U4/U6.U5 tri-snRNP integration but before activation, and have determined the three-dimensional structure of B Delta U1 by single-particle electron cryomicroscopy at a resolution of approximately 40 A. The overall size of the complex is about 370 x 270 x 170 A. The three-dimensional structure features a roughly triangular body linked to a head domain in variable orientations. The body is very similar in size and shape to the isolated U4/U6.U5 tri-snRNP. This provides initial insight into the structural organization of complex B.

Staufen- and FMRP-Containing Neuronal RNPs Are Structurally and Functionally Related to Somatic P Bodies

OpenAIRE

Barbee, Scott A.; Estes, Patricia S.; Cziko, Anne-Marie; Hillebrand, Jens; Luedeman, Rene A.; Coller, Jeff M.; Johnson, Nick; Howlett, Iris C.; Geng, Cuiyun; Ueda, Ryu; Brand, Andrea H.; Newbury, Sarah F.; Wilhelm, James E.; Levine, Richard B.; Nakamura, Akira

2006-01-01

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRPcontaining RNPs in Drosophila neurons contain proteins also present in somatic “P bodies,” including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (ar...

LFQProfiler and RNP(xl): Open-Source Tools for Label-Free Quantification and Protein-RNA Cross-Linking Integrated into Proteome Discoverer.

Science.gov (United States)

Veit, Johannes; Sachsenberg, Timo; Chernev, Aleksandar; Aicheler, Fabian; Urlaub, Henning; Kohlbacher, Oliver

2016-09-02

Modern mass spectrometry setups used in today's proteomics studies generate vast amounts of raw data, calling for highly efficient data processing and analysis tools. Software for analyzing these data is either monolithic (easy to use, but sometimes too rigid) or workflow-driven (easy to customize, but sometimes complex). Thermo Proteome Discoverer (PD) is a powerful software for workflow-driven data analysis in proteomics which, in our eyes, achieves a good trade-off between flexibility and usability. Here, we present two open-source plugins for PD providing additional functionality: LFQProfiler for label-free quantification of peptides and proteins, and RNP(xl) for UV-induced peptide-RNA cross-linking data analysis. LFQProfiler interacts with existing PD nodes for peptide identification and validation and takes care of the entire quantitative part of the workflow. We show that it performs at least on par with other state-of-the-art software solutions for label-free quantification in a recently published benchmark ( Ramus, C.; J. Proteomics 2016 , 132 , 51 - 62 ). The second workflow, RNP(xl), represents the first software solution to date for identification of peptide-RNA cross-links including automatic localization of the cross-links at amino acid resolution and localization scoring. It comes with a customized integrated cross-link fragment spectrum viewer for convenient manual inspection and validation of the results.

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods

DEFF Research Database (Denmark)

Heegaard, N H; Larsen, Martin Røssel; Muncrief, T

2000-01-01

ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein...

Crystallization and preliminary X-ray crystallographic studies of transportin 1 in complex with nucleocytoplasmic shuttling and nuclear localization fragments

International Nuclear Information System (INIS)

Imasaki, Tsuyoshi; Shimizu, Toshiyuki; Hashimoto, Hiroshi; Hidaka, Yuji; Yamada, Michiyuki; Sato, Mamoru

2006-01-01

Transportin 1 was cocrystallized with nucleocytoplasmic shuttling fragments of JKTBP and hnRNP D and a nuclear localization fragment of TAP. X-ray diffraction data were collected using synchrotron radiation at SPring-8. Nucleocytoplasmic transport of proteins with molar masses of larger than 60 000 is mediated by transport receptors. The transport receptor transportin1 (Trn1) transports various kinds of RNA-binding proteins such as JKTBP, hnRNP D and TAP. Trn1 was successfully cocrystallized with nucleocytoplasmic shuttling fragments of JKTBP and hnRNP D and a nuclear localization fragment of TAP. The crystal of the Trn1–JKTBP fragment complex belongs to space group P2 1 2 1 2, with unit-cell parameters a = 131.5, b =171.5, c = 68.2 Å. The crystals of Trn1 in complex with hnRNP D and TAP fragments are orthorhombic, space group P2 1 2 1 2 1 , with unit-cell parameters a = 69.1, b = 119.1, c = 151.1 Å and a = 69.0, b = 119.1, c = 146.0 Å, respectively. The crystals diffracted to beyond 3.0, 3.2 and 2.4 Å resolution, respectively, using synchrotron radiation at SPring-8

Rasputin functions as a positive regulator of orb in Drosophila oogenesis.

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Alexandre Costa

Full Text Available The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. We have previously shown that Rasputin (Rin, the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP, associates with Orb in a messenger ribonucleoprotein (mRNP complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. Here we show that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila.

Rasputin functions as a positive regulator of orb in Drosophila oogenesis.

Science.gov (United States)

Costa, Alexandre; Pazman, Cecilia; Sinsimer, Kristina S; Wong, Li Chin; McLeod, Ian; Yates, John; Haynes, Susan; Schedl, Paul

2013-01-01

The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A) tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1)K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. We have previously shown that Rasputin (Rin), the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP), associates with Orb in a messenger ribonucleoprotein (mRNP) complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. Here we show that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila.

Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E

DEFF Research Database (Denmark)

Fierro-Monti, Ivo; Mohammed, Shabaz; Matthiesen, Rune

2006-01-01

Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins......-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated...... levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP...

RISC assembly: Coordination between small RNAs and Argonaute proteins.

Science.gov (United States)

Kobayashi, Hotaka; Tomari, Yukihide

2016-01-01

Non-coding RNAs generally form ribonucleoprotein (RNP) complexes with their partner proteins to exert their functions. Small RNAs, including microRNAs, small interfering RNAs, and PIWI-interacting RNAs, assemble with Argonaute (Ago) family proteins into the effector complex called RNA-induced silencing complex (RISC), which mediates sequence-specific target gene silencing. RISC assembly is not a simple binding between a small RNA and Ago; rather, it follows an ordered multi-step pathway that requires specific accessory factors. Some steps of RISC assembly and RISC-mediated gene silencing are dependent on or facilitated by particular intracellular platforms, suggesting their spatial regulation. In this review, we summarize the currently known mechanisms for RISC assembly of each small RNA class and propose a revised model for the role of the chaperone machinery in the duplex-initiated RISC assembly pathway. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

UV cross-linking of polypeptides associated with 3'-terminal exons

International Nuclear Information System (INIS)

Stolow, D.T.; Berget, S.M.

1990-01-01

Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

Science.gov (United States)

Aviner, Ranen; Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo; Elroy-Stein, Orna

2017-06-02

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of nuclear accumulation of influenza NP antigen in von Magnus virus-infected cells.

Science.gov (United States)

Maeno, K; Aoki, H; Hamaguchi, M; Iinuma, M; Nagai, Y; Matsumoto, T; Takeura, S; Shibata, M

1981-01-01

When 1-5C-4 cells were infected with von Magnus virus derived from influenza A/RI/5+ virus by successive undiluted passages in chick embryos, virus-specific proteins were synthesized but production of infectious virus was inhibited. In these cells the synthesis of viral RNA was suppressed and the nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to standard virus-infected cells in which the antigen was distributed throughout the whole cell. The intracellular location and migration of NP were determined by isotope labeling and sucrose gradient centrifugation of subcellular fractions. In standard virus-infected cell NP polypeptide was present predominantly in the cytoplasm in the form of viral ribonucleoprotein (RNP) and intranuclear RNP was detected in reduced amounts. In contrast, in von Magnus virus-infected cells NP polypeptide was present predominantly in the nucleus in a nonassembled, soluble from and the amount of cytoplasmic RNP was considerably reduced. After short-pulse labeling NP was detected exclusively in the cytoplasm in a soluble form and after a chase a large proportion of such soluble NP was seen in the nucleus. It is suggested that a large proportion of the NP synthesized in von Magnus virus-infected cells in not assembled into cytoplasmic RNP because of the lack of available RNA and the NP migrated into the nucleus and remained there.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

OpenAIRE

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-01-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic ...

Long noncoding RNA NEAT1 promotes cell proliferation and invasion by regulating hnRNP A2 expression in hepatocellular carcinoma cells

Directory of Open Access Journals (Sweden)

Mang YY

2017-02-01

Full Text Available Yuanyi Mang, Li Li, Jianghua Ran, Shengning Zhang, Jing Liu, Laibang Li, Yiming Chen, Jian Liu, Yang Gao, Gang Ren Department of Hepato-Biliary-Pancreatic Surgery, The Calmette Affiliated Hospital of Kunming Medical University, The First Hospital of Kunming, Kunming, Yunnan, People’s Republic of China Abstract: Growing evidence demonstrates that long noncoding RNAs (lncRNAs are involved in the progression of various cancers, including hepatocellular carcinoma (HCC. The role of nuclear-enriched abundant transcript 1 (NEAT1, an essential lncRNA for the formation of nuclear body paraspeckles, has not been fully explored in HCC. We aimed to determine the expression, roles and functional mechanisms of NEAT1 in the proliferation and invasion of HCC. Based on real-time polymerase chain reaction data, we suggest that NEAT1 is upregulated in HCC tissues compared with noncancerous liver tissues. The knockdown of NEAT1 altered global gene expression patterns and reduced HCC cell proliferation, invasion and migration. RNA immunoprecipitation and RNA pull-down assays confirmed that U2AF65 binds to NEAT1. Furthermore, the study indicated that NEAT1 regulated hnRNP A2 expression and that this regulation may be associated with the NEAT1–U2AF65 protein complex. Thus, the NEAT1-hnRNP A2 regulation mechanism promotes HCC pathogenesis and may provide a potential target for the prognosis and treatment of HCC. Keywords: long noncoding RNA, NEAT1, RNA-binding protein, HCC

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Leder, Verena [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lummer, Martina [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Tegeler, Kathrin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Humpert, Fabian [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lewinski, Martin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Schüttpelz, Mark [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Staiger, Dorothee, E-mail: dorothee.staiger@uni-bielefeld.de [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany)

2014-10-10

Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R{sup 49} abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K{sub d} value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R{sup 49} that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

International Nuclear Information System (INIS)

Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee

2014-01-01

Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R 49 abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K d value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R 49 that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding

Structure of the Hantavirus Nucleoprotein Provides Insights into the Mechanism of RNA Encapsidation

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Daniel Olal

2016-03-01

Full Text Available Hantaviruses are etiological agents of life-threatening hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. The nucleoprotein (N of hantavirus is essential for viral transcription and replication, thus representing an attractive target for therapeutic intervention. We have determined the crystal structure of hantavirus N to 3.2 Å resolution. The structure reveals a two-lobed, mostly α-helical structure that is distantly related to that of orthobunyavirus Ns. A basic RNA binding pocket is located at the intersection between the two lobes. We provide evidence that oligomerization is mediated by amino- and C-terminal arms that bind to the adjacent monomers. Based on these findings, we suggest a model for the oligomeric ribonucleoprotein (RNP complex. Our structure provides mechanistic insights into RNA encapsidation in the genus Hantavirus and constitutes a template for drug discovery efforts aimed at combating hantavirus infections.

An AU-rich element in the 3{prime} untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation

Energy Technology Data Exchange (ETDEWEB)

Chen, Qiuyun; Adams, C.C.; Usack, L. [Cornell Univ., Ithaca, NY (United States)] [and others

1995-04-01

In chloroplasts, the 3{prime} untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3{prime}-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3{prime} IR-containing RNA (3{prime} IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b{sub 6}/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T{sub 1} digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3{prime} IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3{prime} untranslated region, only a single copy, which we have termed box II, appears to be essential for in vivo protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3{prime}-end processing and/or influence the stability of petD mRNA in chloroplasts. 48 refs., 9 figs., 2 tabs.

Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

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Roopa Thapar

2015-05-01

Full Text Available The peptidyl-prolyl cis-trans isomerases (PPIases that include immunophilins (cyclophilins and FKBPs and parvulins (Pin1, Par14, Par17 participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.

In vitro reconstitution of the active T. castaneum telomerase.

Science.gov (United States)

Schuller, Anthony P; Harkisheimer, Michael J; Skordalakes, Emmanuel

2011-07-14

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro. Telomerase is a specialized reverse transcriptase that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit. Additionally, telomerase is dormant in most somatic cells in adults, but is active in cancer cells where it promotes cell immortality. The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres. Prior to 2008, only structures for individual telomerase domains had been solved. A major breakthrough in this field came from the determination of the crystal structure of the active, catalytic subunit of T. castaneum telomerase, TcTERT. Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

Appearance of newly formed mRNA and rRNA as ribonucleoprotein-particles in the cytoplasmic subribosomal fraction of pea embryos

International Nuclear Information System (INIS)

Takahashi, Noribumi; Takaiwa, Fumio; Fukuei, Keisuke; Sakamaki, Tadashi; Tanifuji, Shigeyuki

1977-01-01

Incorporation studies with 3 H-uridine or 3 H-adenosine showed that germinating pea embryos synthesize all types of poly A(+) RNA, rRNA and 4-5S RNA at the early stage of germination. After the pulse labeling for 30 min, only heterodisperse RNA and 4-5S RNA appeared in the cytoplasm as labeled RNA species. At this time the radioactivity was associated with cytoplasmic structures heavier than 80S and RNP particles of 68-70S, 52-55S, 36-38S and 20-22S which are presumed to be free mRNP particles in plants. When the pulse-labeled embryos were incubated for a further 60 min in an isotope-free medium, the labeled 17S and 25S rRNA emerged in the cytoplasm, together with labeled heterodisperse and 4-5S RNAs. More radioactivity accumulated in the regions of the polysome, 62-65S and 38-42S particles. The results of analysis of RNAs extracted from the whole cytoplasm, polysome or subribosomal fractions indicated that small subunits of newly formed ribosomes appear more rapidly in the cytoplasm than new large subunits, which accumulate for a while as free particles in the cytoplasm than are incorporated into polysomes. The actinomycin treatment which caused preferential inhibition of rRNA synthesis reduced the accumulation of free, newly formed ribosome subunits and partially permitted detection of the presumed mRNP particles in the subribosomal region even after the chase treatment. (auth.)

Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation.

Science.gov (United States)

Meyerson, Nicholas R; Zhou, Ligang; Guo, Yusong R; Zhao, Chen; Tao, Yizhi J; Krug, Robert M; Sawyer, Sara L

2017-11-08

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of the catalytic center of the Ebola virus L polymerase.

Science.gov (United States)

Schmidt, Marie Luisa; Hoenen, Thomas

2017-10-01

Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized. Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication. We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential

Characterization of the catalytic center of the Ebola virus L polymerase.

Directory of Open Access Journals (Sweden)

Marie Luisa Schmidt

2017-10-01

Full Text Available Ebola virus (EBOV causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp of L, which is one of the most promising molecular targets, has never been experimentally characterized.Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication.We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target

Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition.

Science.gov (United States)

Lostalé-Seijo, Irene; Louzao, Iria; Juanes, Marisa; Montenegro, Javier

2017-12-01

The discovery of RNA guided endonucleases has emerged as one of the most important tools for gene edition and biotechnology. The selectivity and simplicity of the CRISPR/Cas9 strategy allows the straightforward targeting and editing of particular loci in the cell genome without the requirement of protein engineering. However, the transfection of plasmids encoding the Cas9 and the guide RNA could lead to undesired permanent recombination and immunogenic responses. Therefore, the direct delivery of transient Cas9 ribonucleoprotein constitutes an advantageous strategy for gene edition and other potential therapeutic applications of the CRISPR/Cas9 system. The covalent fusion of Cas9 with penetrating peptides requires multiple incubation steps with the target cells to achieve efficient levels of gene edition. These and other recent reports suggested that covalent conjugation of the anionic Cas9 ribonucleoprotein to cationic peptides would be associated with a hindered nuclease activity due to undesired electrostatic interactions. We here report a supramolecular strategy for the direct delivery of Cas9 by an amphiphilic penetrating peptide that was prepared by a hydrazone bond formation between a cationic peptide scaffold and a hydrophobic aldehyde tail. The peptide/protein non-covalent nanoparticles performed with similar efficiency and less toxicity than one of the best methods described to date. To the best of our knowledge this report constitutes the first supramolecular strategy for the direct delivery of Cas9 using a penetrating peptide vehicle. The results reported here confirmed that peptide amphiphilic vectors can deliver Cas9 in a single incubation step, with good efficiency and low toxicity. This work will encourage the search and development of conceptually new synthetic systems for transitory endonucleases direct delivery.

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Science.gov (United States)

Subramanian, A; Ranganathan, P; Diamond, S L

1999-09-01

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

Nuclear Export of Messenger RNA

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Jun Katahira

2015-03-01

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

Identification of Neuregulin-2 as a novel stress granule component.

Science.gov (United States)

Kim, Jin Ah; Jayabalan, Aravinth Kumar; Kothandan, Vinoth Kumar; Mariappan, Ramesh; Kee, Younghoon; Ohn, Takbum

2016-08-01

Stress Granules (SGs) are microscopically visible, phase dense aggregates of translationally stalled messenger ribonucleoprotein (mRNP) complexes formed in response to distinct stress conditions. It is generally considered that SG formation is induced to protect cells from conditions of stress. The precise constituents of SGs and the mechanism through which SGs are dynamically regulated in response to stress are not completely understood. Hence, it is important to identify proteins which regulate SG assembly and disassembly. In the present study, we report Neuregulin-2 (NRG2) as a novel component of SGs; furthermore, depletion of NRG2 potently inhibits SG formation. We also demonstrate that NRG2 specifically localizes to SGs under various stress conditions. Knockdown of NRG2 has no effect on stress-induced polysome disassembly, suggesting that the component does not influence early step of SG formation. It was also observed that reduced expression of NRG2 led to marginal increase in cell survival under arsenite-induced stress. [BMB Reports 2016; 49(8): 449-454].

Nuclear Export of Messenger RNA

Science.gov (United States)

Katahira, Jun

2015-01-01

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

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

Science.gov (United States)

Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

2015-01-01

Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

The hnRNP 2H9 gene, which is involved in the splicing reaction, is a multiply spliced gene

DEFF Research Database (Denmark)

Honoré, B

2000-01-01

The hnRNP 2H9 gene products are involved in the splicing process and participate in early heat shock-induced splicing arrest. By combining low/high stringency hybridisation, database search, Northern and Western blotting it is shown that the gene is alternatively spliced into at least six...

Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

DEFF Research Database (Denmark)

Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard

2008-01-01

CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild...... irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs. CONCLUSION: Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non...

Characterization of a viral phosphoprotein binding site on the surface of the respiratory syncytial nucleoprotein.

Science.gov (United States)

Galloux, Marie; Tarus, Bogdan; Blazevic, Ilfad; Fix, Jenna; Duquerroy, Stéphane; Eléouët, Jean-François

2012-08-01

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P(CTD)) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N(NTD)) as a P binding domain. A biochemical characterization of the P(CTD) and molecular modeling of the N(NTD) allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P(CTD) interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

RNA integrity as a quality indicator during the first steps of RNP purifications : A comparison of yeast lysis methods

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Jansen Ralf-Peter

2004-10-01

Full Text Available Abstract Background The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor. Results We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins. Conclusion We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

Science.gov (United States)

Hawse, William F; Boggess, William C; Morel, Penelope A

2017-07-15

The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

In vivo kinetics of U4/U6.U5 tri-snRNP formation in Cajal bodies

Czech Academy of Sciences Publication Activity Database

Novotný, Ivan; Blažíková, Michaela; Staněk, David; Heřman, P.; Malínský, Jan

2011-01-01

Roč. 22, č. 4 (2011), s. 513-523 ISSN 1059-1524 R&D Projects: GA ČR GA204/07/0133; GA AV ČR KAN200520801 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : small nuclear ribonucleoprotein * body components * dynamics Subject RIV: EB - Genetics ; Molecular Biology Impact factor : 4.942, year: 2011

Hepatoma-derived growth factor and nucleolin exist in the same ribonucleoprotein complex

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Bremer Stephanie

2013-01-01

Full Text Available Abstract Background Hepatoma-derived growth factor (HDGF is a protein which is highly expressed in a variety of tumours. HDGF has mitogenic, angiogenic, neurotrophic and antiapoptotic activity but the molecular mechanisms by which it exerts these activities are largely unknown nor has its biological function in tumours been elucidated. Mass spectrometry was performed to analyse the HDGFStrep-tag interactome. By Pull–down-experiments using different protein and nucleic acid constructs the interaction of HDGF and nucleolin was investigated further. Results A number of HDGFStrep-tag copurifying proteins were identified which interact with RNA or are involved in the cellular DNA repair machinery. The most abundant protein, however, copurifying with HDGF in this approach was nucleolin. Therefore we focus on the characterization of the interaction of HDGF and nucleolin in this study. We show that expression of a cytosolic variant of HDGF causes a redistribution of nucleolin into the cytoplasm. Furthermore, formation of HDGF/nucleolin complexes depends on bcl-2 mRNA. Overexpression of full length bcl-2 mRNA increases the number of HDGF/nucleolin complexes whereas expression of only the bcl-2 coding sequence abolishes interaction completely. Further examination reveals that the coding sequence of bcl-2 mRNA together with either the 5′ or 3′ UTR is sufficient for formation of HDGF/nucleolin complexes. When bcl-2 coding sequence within the full length cDNA is replaced by a sequence coding for secretory alkaline phosphatase complex formation is not enhanced. Conclusion The results provide evidence for the existence of HDGF and nucleolin containing nucleoprotein complexes which formation depends on the presence of specific mRNAs. The nature of these RNAs and other components of the complexes should be investigated in future.

Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1

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Ren, Yi [Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States; Schmiege, Philip [Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States; Blobel, Günter [Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States

2017-01-06

mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2’s intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1.

Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1.

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Ren, Yi; Schmiege, Philip; Blobel, Günter

2017-01-06

mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2's intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1.

Rings in the neonate.

LENUS (Irish Health Repository)

Hackett, C B

2011-02-01

Neonatal lupus erythematosus (NLE) is an uncommon disease of the neonate. It is believed to be caused by the transplacental passage of maternal autoantibodies to the ribonucleoproteins (Ro\\/SSA, La\\/SSB or rarely U RNP) as these are almost invariably present in NLE sera. The most common clinical manifestations include cutaneous lupus lesions and congenital complete heart block. Hepatobiliary and haematologic abnormalities are reported less frequently. We describe a patient with cutaneous NLE to illustrate and raise awareness of the characteristic annular eruption of this condition. We also emphasize the need for thorough investigation for concomitant organ involvement and for maternal education regarding risk in future pregnancies.

Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

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Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

2014-01-01

Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

Novel redox nanomedicine improves gene expression of polyion complex vector

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Kazuko Toh, Toru Yoshitomi, Yutaka Ikeda and Yukio Nagasaki

2011-01-01

Full Text Available Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP as an ROS scavenger. When polyethyleneimine (PEI/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

Convergent evolution of germ granule nucleators: A hypothesis.

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Kulkarni, Arpita; Extavour, Cassandra G

2017-10-01

Germ cells have been considered "the ultimate stem cell" because they alone, during normal development of sexually reproducing organisms, are able to give rise to all organismal cell types. Morphological descriptions of a specialized cytoplasm termed 'germ plasm' and associated electron dense ribonucleoprotein (RNP) structures called 'germ granules' within germ cells date back as early as the 1800s. Both germ plasm and germ granules are implicated in germ line specification across metazoans. However, at a molecular level, little is currently understood about the molecular mechanisms that assemble these entities in germ cells. The discovery that in some animals, the gene products of a small number of lineage-specific genes initiate the assembly (also termed nucleation) of germ granules and/or germ plasm is the first step towards facilitating a better understanding of these complex biological processes. Here, we draw on research spanning over 100years that supports the hypothesis that these nucleator genes may have evolved convergently, allowing them to perform analogous roles across animal lineages. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Coilin phosphorylation mediates interaction with SMN and SmB′

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Toyota, Cory G.; Davis, Misty D.; Cosman, Angela M.; Hebert, Michael D.

2010-01-01

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB. PMID:19997741

Coilin phosphorylation mediates interaction with SMN and SmB'.

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Toyota, Cory G; Davis, Misty D; Cosman, Angela M; Hebert, Michael D

2010-04-01

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB' binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB' than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins.

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Liang, Zhen; Chen, Kunling; Zhang, Yi; Liu, Jinxing; Yin, Kangquan; Qiu, Jin-Long; Gao, Caixia

2018-03-01

This protocol is an extension to: Nat. Protoc. 9, 2395-2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment. This protocol serves as an extension of our previously published protocol on genome editing in bread wheat using CRISPR/Cas9 plasmids delivered by particle bombardment. The methods we describe not only eliminate random integration of CRISPR/Cas9 into genomic DNA, but also reduce off-target effects. In this protocol extension article, we present detailed protocols for preparation of IVTs and RNPs; validation by PCR/restriction enzyme (RE) and next-generation sequencing; delivery by biolistics; and recovery of mutants and identification of mutants by pooling methods and Sanger sequencing. To use these protocols, researchers should have basic skills and experience in molecular biology and biolistic transformation. By using these protocols, plants edited without the use of any foreign DNA can be generated and identified within 9-11 weeks.

Endogenous spar tin, mutated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons

International Nuclear Information System (INIS)

Robay, Dimitri; Patel, Heema; Simpson, Michael A.; Brown, Nigel A.; Crosby, Andrew H.

2006-01-01

Mutation of spartin (SPG20) underlies a complicated form of hereditary spastic paraplegia, a disorder principally defined by the degeneration of upper motor neurons. Using a polyclonal antibody against spartin to gain insight into the function of the endogenous molecule, we show that the endogenous molecule is present in two main isoforms of 85 kDa and 100 kDa, and 75 kDa and 85 kDa in human and murine, respectively, with restricted subcellular localization. Immunohistochemical studies on human and mouse embryo sections and in vitro cell studies indicate that spartin is likely to possess both nuclear and cytoplasmic functions. The nuclear expression of spartin closely mirrors that of the snRNP (small nuclear ribonucleoprotein) marker α-Sm, a component of the spliceosome. Spartin is also enriched at the centrosome within mitotic structures. Notably we show that spartin protein undergoes dynamic positional changes in differentiating human SH-SY5Y cells. In undifferentiated non-neuronal cells, spartin displays a nuclear and diffuse cytosolic profile, whereas spartin transiently accumulates in the trans-Golgi network and subsequently decorates discrete puncta along neurites in terminally differentiated neuroblastic cells. Investigation of these spartin-positive vesicles reveals that a large proportion colocalizes with the synaptic vesicle marker synaptotagmin. Spartin is also enriched in synaptic-like structures and in synaptic vesicle-enriched fraction

RNA Binding Proteins in Eye Development and Disease: Implication of Conserved RNA Granule Components

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Dash, Soma; Siddam, Archana D.; Barnum, Carrie E.; Janga, Sarath Chandra

2016-01-01

The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1 and Bmp4 are commonly required for their development. In contrast, our understanding of post-transcriptional regulation in eye development and disease, particularly regarding the function of RNA binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila, as well as several vertebrate models such as fish, frog, chicken and mouse. Interestingly, of the 42 RBPs that have been investigated with for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as Processing bodies (P-bodies), Stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate post-transcriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2 and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving post-transcriptional regulatory networks in eye development and disease. PMID:27133484

Morphogenesis of the infectious HIV-1 virion

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Jun-Ichi eSakuragi

2011-12-01

Full Text Available The virion of HIV-1 is spherical and viral glycoprotein spikes (gp120, gp41 protrude from its envelope. The characteristic cone-shaped core exists within the virion, caging the ribonucleoprotein (RNP complex, which is comprised of viral RNA, nucleocapsid (NC and viral enzymes. The HIV-1 virion is budded and released from the infected cell as an immature donut-shaped particle. During or immediately after release, viral protease (PR is activated and subsequently processes the viral structural protein Gag. Through this maturation process, virions acquire infectivity, but its mechanism and transition of morphology largely remain unclear. Recent technological advances in experimental devices and techniques have made it possible to closely dissect the viral production site on the cell, the exterior – or even the interior – of an individual virion, and many new aspects on virion morphology and maturation. In this manuscript, I review the morphogenesis of HIV-1 virions. I focus on several studies, including some of our recent findings, which examined virion formation and/or maturation processes. The story of novel compound, which inhibits virion maturation, and the importance of maturation research are also discussed.

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

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Ki-Eun Park

2016-05-01

Full Text Available 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.

Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles

International Nuclear Information System (INIS)

Kroemer, A.

1987-01-01

Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ribonucleoprotein particles U1 to U6, since they are specifically bound by anti-Sm antibodies. These criteria characterize the RNAs that are 145 and 66/65 nucleotides in length as U-type snRNAs. Upon gel filtration, the RNAs are found within particles of molecular weights ≅ 150,000 and 115,000 respectively. The RNA of 145 nucleotides represents a different minor snRNA, designated U11, whereas the RNA of 66/65 nucleotides may correspond to either mammalian U7 or U10 RNA

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate.

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Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

2013-08-01

Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.

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

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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. Copyright © 2016 Elsevier Inc. All rights reserved.

Plant RNA Regulatory Network and RNA Granules in Virus Infection

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Kristiina Mäkinen

2017-12-01

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

Plant RNA Regulatory Network and RNA Granules in Virus Infection.

Science.gov (United States)

Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

2017-01-01

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

CYTOCHEMICAL STUDIES OF THE NUCLEOPROTEINS OF HELA CELLS INFECTED WITH HERPES VIRUS.

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Love, R; Wildy, P

1963-05-01

The morphological and cytochemical changes in HeLa cells infected with herpes virus have been studied at frequent intervals during infection and related to the growth of virus and the multiplicity of the virus inoculum. Infection with a high multiplicity inoculum produced enlargement and extrusion of small ribonucleoprotein (RNP) bodies in the nucleoli (nucleolini) to form RNP bodies in the nucleoplasm (B bodies) beginning (1/2) hour after infection. 3 hours after infection, RNP of the pars amorpha appeared to diffuse into the adjacent nucleoplasm, where, (1/2) hour later, the classical type A inclusion or A body first appeared. The A bodies displaced the B bodies and the nucleoli and eventually filled the nucleus. 6 hours after infection, minute granules containing RNA, DNA, and non-histone protein appeared inside the A bodies (A granules) and increased in number until the late stages of infection, when they disappeared. 18 hours after infection, at the time when the A bodies came to fill the nucleus completely, extrusion of RNP from the nucleus produced cytoplasmic masses which have been termed C bodies. B bodies were formed in the majority of cells before the maturation of infectious virus, but the number of B bodies could not be correlated with the amount of virus in the cell or with the multiplicity of the inoculum. It is suggested that the formation of B bodies may be the result of inhibition of the onset of mitotic division by a mechanism which does not inhibit the formation of RNA in the nucleolini. The nature of the A bodies, the A granules, and the C bodies is discussed and it is concluded that the A granules may represent aggregations of maturing virus in the nucleus. The progression of some C mitotic metaphases to the formation of post-C mitotic multinucleated giant cells is described. These are distinct from syncytia formed by cell fusion.

Supraspliceosomes at Defined Functional States Portray the Pre-Assembled Nature of the Pre-mRNA Processing Machine in the Cell Nucleus

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Hani Kotzer-Nevo

2014-06-01

Full Text Available When isolated from mammalian cell nuclei, all nuclear pre-mRNAs are packaged in multi-subunit large ribonucleoprotein complexes—supraspliceosomes—composed of four native spliceosomes interconnected by the pre-mRNA. Supraspliceosomes contain all five spliceosomal U snRNPs, together with other splicing factors, and are functional in splicing. Supraspliceosomes studied thus far represent the steady-state population of nuclear pre-mRNAs that were isolated at different stages of the splicing reaction. To analyze specific splicing complexes, here, we affinity purified Pseudomonas aeruginosa phage 7 (PP7-tagged splicing complexes assembled in vivo on Adenovirus Major Late (AdML transcripts at specific functional stages, and characterized them using molecular techniques including mass spectrometry. First, we show that these affinity purified splicing complexes assembled on PP7-tagged AdML mRNA or on PP7-tagged AdML pre-mRNA are assembled in supraspliceosomes. Second, similar to the general population of supraspliceosomes, these defined supraspliceosomes populations are assembled with all five U snRNPs at all splicing stages. This study shows that dynamic changes in base-pairing interactions of U snRNA:U snRNA and U snRNA:pre-mRNA that occur in vivo during the splicing reaction do not require changes in U snRNP composition of the supraspliceosome. Furthermore, there is no need to reassemble a native spliceosome for the splicing of each intron, and rearrangements of the interactions will suffice.

Human Ro60 (SSA2) genomic organization and sequence alterations, examined in cutaneous lupus erythematosus.

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Millard, T P; Ashton, G H S; Kondeatis, E; Vaughan, R W; Hughes, G R V; Khamashta, M A; Hawk, J L M; McGregor, J M; McGrath, J A

2002-02-01

The Ro 60 kDa protein (Ro60 or SSA2) is the major component of the Ro ribonucleoprotein (Ro RNP) complex, to which an immune response is a specific feature of several autoimmune diseases. The genomic organization and any sequence variation within the DNA encoding Ro60 are unknown. To characterize the Ro60 gene structure and to assess whether any sequence alterations might be associated with serum anti-Ro antibody in subacute cutaneous lupus erythematosus (SCLE), thus potentially providing new insight into disease pathogenesis. The cDNA sequence for Ro60 was obtained from the NCBI database and used for a BLAST search for a clone containing the entire genomic sequence. The intron-exon borders were confirmed by designing intronic primer pairs to flank each exon, which were then used to amplify genomic DNA for automated sequencing from 36 caucasian patients with SCLE (anti-Ro positive) and 49 with discoid LE (DLE, anti-Ro negative), in addition to 36 healthy caucasian controls. Heteroduplex analysis of polymerase chain reaction (PCR) products from patients and controls spanning all Ro60 exons (1-8) revealed a common bandshift in the PCR products spanning exon 7. Sequencing of the corresponding PCR products demonstrated an A > G substitution at nucleotide position 1318-7, within the consensus acceptor splice site of exon 7 (GenBank XM001901). The allele frequencies were major allele A (0.71) and minor allele G (0.29) in 72 control chromosomes, with no significant differences found between SCLE patients, DLE patients and controls. The genomic organization of the DNA encoding the Ro60 protein is described, including a common polymorphism within the consensus acceptor splice site of exon 7. Our delineation of a strategy for the genomic amplification of Ro60 forms a basis for further examination of the pathological functions of the Ro RNP in autoimmune disease.

Interaction of influenza virus proteins with nucleosomes

International Nuclear Information System (INIS)

Garcia-Robles, Inmaculada; Akarsu, Hatice; Mueller, Christoph W.; Ruigrok, Rob W.H.; Baudin, Florence

2005-01-01

During influenza virus infection, transcription and replication of the viral RNA take place in the cell nucleus. Directly after entry in the nucleus the viral ribonucleoproteins (RNPs, the viral subunits containing vRNA, nucleoprotein and the viral polymerase) are tightly associated with the nuclear matrix. Here, we have analysed the binding of RNPs, M1 and NS2/NEP proteins to purified nucleosomes, reconstituted histone octamers and purified single histones. RNPs and M1 both bind to the chromatin components but at two different sites, RNP to the histone tails and M1 to the globular domain of the histone octamer. NS2/NEP did not bind to nucleosomes at all. The possible consequences of these findings for nuclear release of newly made RNPs and for other processes during the infection cycle are discussed

Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

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Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

2010-10-15

Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

Structural and Functional Motifs in Influenza Virus RNAs

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Damien Ferhadian

2018-03-01

Full Text Available Influenza A viruses (IAV are responsible for recurrent influenza epidemics and occasional devastating pandemics in humans and animals. They belong to the Orthomyxoviridae family and their genome consists of eight (- sense viral RNA (vRNA segments of different lengths coding for at least 11 viral proteins. A heterotrimeric polymerase complex is bound to the promoter consisting of the 13 5′-terminal and 12 3′-terminal nucleotides of each vRNA, while internal parts of the vRNAs are associated with multiple copies of the viral nucleoprotein (NP, thus forming ribonucleoproteins (vRNP. Transcription and replication of vRNAs result in viral mRNAs (vmRNAs and complementary RNAs (cRNAs, respectively. Complementary RNAs are the exact positive copies of vRNAs; they also form ribonucleoproteins (cRNPs and are intermediate templates in the vRNA amplification process. On the contrary, vmRNAs have a 5′ cap snatched from cellular mRNAs and a 3′ polyA tail, both gained by the viral polymerase complex. Hence, unlike vRNAs and cRNAs, vmRNAs do not have a terminal promoter able to recruit the viral polymerase. Furthermore, synthesis of at least two viral proteins requires vmRNA splicing. Except for extensive analysis of the viral promoter structure and function and a few, mostly bioinformatics, studies addressing the vRNA and vmRNA structure, structural studies of the influenza A vRNAs, cRNAs, and vmRNAs are still in their infancy. The recent crystal structures of the influenza polymerase heterotrimeric complex drastically improved our understanding of the replication and transcription processes. The vRNA structure has been mainly studied in vitro using RNA probing, but its structure has been very recently studied within native vRNPs using crosslinking and RNA probing coupled to next generation RNA sequencing. Concerning vmRNAs, most studies focused on the segment M and NS splice sites and several structures initially predicted by bioinformatics analysis

Heterogeneous nuclear ribonuclear protein K interacts with Sindbis virus nonstructural proteins and viral subgenomic mRNA

International Nuclear Information System (INIS)

Burnham, Andrew J.; Gong, Lei; Hardy, Richard W.

2007-01-01

Alphaviruses are a group of arthropod-borne human and animal pathogens that can cause epidemics of significant public health and economic consequence. Alphavirus RNA synthesis requires four virally encoded nonstructural proteins and probably a number of cellular proteins. Using comparative two-dimensional electrophoresis we were able to identify proteins enriched in cytoplasmic membrane fractions containing viral RNA synthetic complexes following infection with Sindbis virus. Our studies demonstrated the following: (i) the host protein hnRNP K is enriched in cytoplasmic membrane fractions following Sindbis virus infection, (ii) viral nonstructural proteins co-immunoprecipitate with hnRNP K, (iii) nsP2 and hnRNP K co-localize in the cytoplasm of Sindbis virus infected cells, (iv) Sindbis virus subgenomic mRNA, but not genomic RNA co-immunoprecipitates with hnRNP K, (v) viral RNA does not appear to be required for the interaction of hnRNP K with the nonstructural proteins. Potential functions of hnRNP K during virus replication are discussed

Crystal Structure of a CRISPR RNA-guided Surveillance Complex Bound to a ssDNA Target

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Mulepati, Sabin [Johns Hopkins Univ., Baltimore, MD (United States); Heroux, Annie; Bailey, Scott [Johns Hopkins Univ., Baltimore, MD (United States)

2014-09-19

In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

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

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Goldfarb, Katherine C; Cech, Thomas R

2017-01-01

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

The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition.

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Onofrillo, Carmine; Galbiati, Alice; Montanaro, Lorenzo; Derenzini, Massimo

2017-01-17

Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA.

Knocking Down Snrnp200 Initiates Demorphogenesis of Rod Photoreceptors in Zebrafish

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Yuan Liu

2015-01-01

Full Text Available Purpose. The small nuclear ribonucleoprotein 200 kDa (SNRNP200 gene is a fundamental component for precursor message RNA (pre-mRNA splicing and has been implicated in the etiology of autosomal dominant retinitis pigmentosa (adRP. This study aims to determine the consequences of knocking down Snrnp200 in zebrafish. Methods. Expression of the Snrnp200 transcript in zebrafish was determined via whole mount in situ hybridization. Morpholino oligonucleotide (MO aiming to knock down the expression of Snrnp200 was injected into zebrafish embryos, followed by analyses of aberrant splicing and expression of the U4/U6-U5 tri-small nuclear ribonucleoproteins (snRNPs components and retina-specific transcripts. Systemic changes and retinal phenotypes were further characterized by histological study and immunofluorescence staining. Results. Snrnp200 was ubiquitously expressed in zebrafish. Knocking down Snrnp200 in zebrafish triggered aberrant splicing of the cbln1 gene, upregulation of other U4/U6-U5 tri-snRNP components, and downregulation of a panel of retina-specific transcripts. Systemic defects were found correlated with knockdown of Snrnp200 in zebrafish. Only demorphogenesis of rod photoreceptors was detected in the initial stage, mimicking the disease characteristics of RP. Conclusions. We conclude that knocking down Snrnp200 in zebrafish could alter regular splicing and expression of a panel of genes, which may eventually trigger rod defects.

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

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Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

2016-01-01

Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

RNase MRP cleaves pre-tRNASer-Met in the tRNA maturation pathway.

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Saito, Yuichiro; Takeda, Jun; Adachi, Kousuke; Nobe, Yuko; Kobayashi, Junya; Hirota, Kouji; Oliveira, Douglas V; Taoka, Masato; Isobe, Toshiaki

2014-01-01

Ribonuclease mitochondrial RNA processing (RNase MRP) is a multifunctional ribonucleoprotein (RNP) complex that is involved in the maturation of various types of RNA including ribosomal RNA. RNase MRP consists of a potential catalytic RNA and several protein components, all of which are required for cell viability. We show here that the temperature-sensitive mutant of rmp1, the gene for a unique protein component of RNase MRP, accumulates the dimeric tRNA precursor, pre-tRNA(Ser-Met). To examine whether RNase MRP mediates tRNA maturation, we purified the RNase MRP holoenzyme from the fission yeast Schizosaccharomyces pombe and found that the enzyme directly and selectively cleaves pre-tRNA(Ser-Met), suggesting that RNase MRP participates in the maturation of specific tRNA in vivo. In addition, mass spectrometry-based ribonucleoproteomic analysis demonstrated that this RNase MRP consists of one RNA molecule and 11 protein components, including a previously unknown component Rpl701. Notably, limited nucleolysis of RNase MRP generated an active catalytic core consisting of partial mrp1 RNA fragments, which constitute "Domain 1" in the secondary structure of RNase MRP, and 8 proteins. Thus, the present study provides new insight into the structure and function of RNase MRP.

Cross- and Co-Packaging of Retroviral RNAs and Their Consequences

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Lizna M. Ali

2016-10-01

Full Text Available Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins. Moreover, several retroviruses have been shown to have the ability to cross-/co-package gRNA from other retroviruses, despite little sequence homology. This review will compare the determinants of gRNA encapsidation among different retroviruses, followed by an examination of our current understanding of the interaction between diverse viral genomes and heterologous proteins, leading to their cross-/co-packaging. Retroviruses are well-known serious animal and human pathogens, and such a cross-/co-packaging phenomenon could result in the generation of novel viral variants with unknown pathogenic potential. At the same time, however, an enhanced understanding of the molecular mechanisms involved in these specific interactions makes retroviruses an attractive target for anti-viral drugs, vaccines, and vectors for human gene therapy.

The serological response of young dogs to the Flury LEP strain of rabies virus vaccine.

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Aghomo, H O; Oduye, O O; Rupprecht, C E

1990-01-01

The serological response of puppies from Nigeria to live Flury low egg passage (LEP) rabies vaccine was determined. Two sets of puppies were used: one set from rabies-vaccinated bitches and another set from non-vaccinated bitches. Puppies were vaccinated intramuscularly with Flury LEP strain rabies vaccine and serially bled from the 4th week to the 30th week. Serum rabies virus neutralizing antibodies (VNA) were measured by a modified rapid fluorescent focus inhibition test (RFFIT). Puppies from non-vaccinated bitches responded well to vaccination after the 4th week and through to the 10th week of age, showing a progressive increase in VNA. In contrast, puppies from vaccinated bitches responded well to rabies vaccination only at 10 weeks of age, although detectable maternal rabies VNA and rabies anti-ribonucleoprotein (RNP) antibodies had decreased by 6 weeks post partum.

Generation of Recombinant Ebola Viruses Using Reverse Genetics.

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Groseth, Allison

2017-01-01

Reverse genetics systems encompass a wide array of tools aimed at recapitulating some or all of the virus life cycle. In their most complete form, full-length clone systems allow us to use plasmid-encoded versions of the ribonucleoprotein (RNP) components to initiate the transcription and replication of a plasmid-encoded version of the complete viral genome, thereby initiating the complete virus life cycle and resulting in infectious virus. As such this approach is ideal for the generation of tailor-made recombinant filoviruses, which can be used to study virus biology. In addition, the generation of tagged and particularly fluorescent or luminescent viruses can be applied as tools for both diagnostic applications and for screening to identify novel countermeasures. Here we describe the generation and basic characterization of recombinant Ebola viruses rescued from cloned cDNA using a T7-driven system.

Localisation of RNAs into the germ plasm of vitellogenic Xenopus oocytes.

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Sarbjit Nijjar

Full Text Available We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2, we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the "late", Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others.

Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export.

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Krebber, H; Taura, T; Lee, M S; Silver, P A

1999-08-01

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay

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Zavolan Mihaela

2010-10-01

Full Text Available Abstract Background In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate. Results In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs. Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms. Conclusions Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.

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

International Nuclear Information System (INIS)

Chapman, C.P.

1986-01-01

Extracts of sporangiospores of Mucor racemosus contained RNA that readily hybridized with [ 3 H]polyuridylic acid. Prior to germination, this RNA was in a form sedimenting at 31 P-orthophosphate or L-[ 32 S]methionine, absorbance at 280 nm, or hybridization of [ 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

Yeast Interacting Proteins Database: YGR013W, YKL012W [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available tion U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the formation of the second commitm... PRP40 U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the form...ation of the second commitment complex Rows with this prey as prey (1) Rows with

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

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Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

2017-08-21

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

All Small Nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP Localize to Nucleoli; Identification of the Nucleolar Localization Element of U6 snRNA

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Gerbi, Susan A.; Lange, Thilo Sascha

2002-01-01

Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3′ end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3′-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3′ hydroxyl of U6 snRNA to a 3′ phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies. PMID:12221120

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

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Morin, Benjamin; Liang, Bo; Gardner, Erica; Ross, Robin A; Whelan, Sean P J

2017-01-01

We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L

RNA Encapsidation and Packaging in the Phleboviruses

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Katherine E. Hornak

2016-07-01

Full Text Available The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV, severe fever with thrombocytopenia syndrome virus (SFTSV, Uukuniemi virus (UUKV, and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5–7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA are synthesized. The interaction between the vRNA and the viral nucleocapsid (N protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses.

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

International Nuclear Information System (INIS)

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

2008-01-01

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-1β-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1β-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

The directionality of the nuclear transport of the influenza A genome is driven by selective exposure of nuclear localization sequences on nucleoprotein

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Panté Nelly

2009-06-01

Full Text Available Abstract Background Early in infection, the genome of the influenza A virus, consisting of eight complexes of RNA and proteins (termed viral ribonucleoproteins; vRNPs, enters the nucleus of infected cells for replication. Incoming vRNPs are imported into the nucleus of infected cells using at least two nuclear localization sequences on nucleoprotein (NP; NLS1 at the N terminus, and NLS2 in the middle of the protein. Progeny vRNP assembly occurs in the nucleus, and later in infection, these are exported from the nucleus to the cytoplasm. Nuclear-exported vRNPs are different from incoming vRNPs in that they are prevented from re-entering the nucleus. Why nuclear-exported vRNPs do not re-enter the nucleus is unknown. Results To test our hypothesis that the exposure of NLSs on the vRNP regulates the directionality of the nuclear transport of the influenza vRNPs, we immunolabeled the two NLSs of NP (NLS1 and NLS2 and analyzed their surface accessibility in cells infected with the influenza A virus. We found that the NLS1 epitope on NP was exposed throughout the infected cells, but the NLS2 epitope on NP was only exposed in the nucleus of the infected cells. Addition of the nuclear export inhibitor leptomycin B further revealed that NLS1 is no longer exposed in cytoplasmic NP and vRNPs that have already undergone nuclear export. Similar immunolabeling studies in the presence of leptomycin B and with cells transfected with the cDNA of NP revealed that the NLS1 on NP is hidden in nuclear exported-NP. Conclusion NLS1 mediates the nuclear import of newly-synthesized NP and incoming vRNPs. This NLS becomes hidden on nuclear-exported NP and nuclear-exported vRNPs. Thus the selective exposure of the NLS1 constitutes a critical mechanism to regulate the directionality of the nuclear transport of vRNPs during the influenza A viral life cycle.

Intracellular coordination of potyviral RNA functions in infection

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Kristiina eMäkinen

2014-03-01

Full Text Available Abstract Establishment of an infection cycle requires mechanisms to allocate the genomes of (+-stranded RNA viruses in a balanced ratio to translation, replication, encapsidation, and movement, as well as mechanisms to prevent translocation of viral RNA (vRNA to cellular RNA degradation pathways. The ratio of vRNA allocated to various functions is likely balanced by the availability of regulatory proteins or competition of the interaction sites within regulatory ribonucleoprotein (RNP complexes. Due to the transient nature of viral processes and the interdependency between vRNA pathways, it is technically demanding to work out the exact molecular mechanisms underlying vRNA regulation. A substantial number of viral and host proteins have been identified that facilitate the steps that lead to the assembly of a functional potyviral RNA replication complex and their fusion with chloroplasts. Simultaneously with on-going viral replication, part of the replicated potyviral RNA enters movement pathways. Although not much is known about the processes of potyviral RNA release from viral replication complexes (VRCs, the molecular interactions involved in these processes determine the fate of the replicated vRNA. Some viral and host cell proteins have been described that direct replicated potyviral RNA to translation to enable potyviral gene expression and productive infection. The antiviral defense of the cell causes vRNA degradation by RNA silencing. We hypothesize that also plant pathways involved in mRNA decay may have a role in the coordination of potyviral RNA expression. In this review, we discuss the roles of different potyviral and host proteins in the coordination of various potyviral RNA functions.

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

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

2017-04-01

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

Hormone response element binding proteins: novel regulators of vitamin D and estrogen signaling.

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Lisse, Thomas S; Hewison, Martin; Adams, John S

2011-03-01

Insights from vitamin D-resistant New World primates and their human homologues as models of natural and pathological insensitivity to sterol/steroid action have uncovered a family of novel intracellular vitamin D and estrogen regulatory proteins involved in hormone action. The proteins, known as "vitamin D or estrogen response element-binding proteins", behave as potent cis-acting, transdominant regulators to inhibit steroid receptor binding to DNA response elements and is responsible for vitamin D and estrogen resistances. This set of interactors belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family of previously known pre-mRNA-interacting proteins. This review provides new insights into the mechanism by which these novel regulators of signaling and metabolism can act to regulate responses to vitamin D and estrogen. In addition the review also describes other molecules that are known to influence nuclear receptor signaling through interaction with hormone response elements. Copyright © 2011 Elsevier Inc. All rights reserved.

U bodies respond to nutrient stress in Drosophila

International Nuclear Information System (INIS)

Buckingham, Mickey; Liu, Ji-Long

2011-01-01

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by mutation of the survival motor neuron 1 (SMN1) gene. Cytoplasmic SMN protein-containing granules, known as U snRNP bodies (U bodies), are thought to be responsible for the assembly and storage of small nuclear ribonucleoproteins (snRNPs) which are essential for pre-mRNA splicing. U bodies exhibit close association with cytoplasmic processing bodies (P bodies), which are involved in mRNA decay and translational repression. The close association of the U body and P body in Drosophila resemble that of the stress granule and P body in yeast and mammalian cells. However, it is unknown whether the U body is responsive to any stress. Using Drosophila oogenesis as a model, here we show that U bodies increase in size following nutritional deprivation. Despite nutritional stress, U bodies maintain their close association with P bodies. Our results show that U bodies are responsive to nutrition changes, presumably through the U body–P body pathway.

Splicing regulatory factors, ageing and age-related disease.

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Latorre, Eva; Harries, Lorna W

2017-07-01

Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease. Copyright © 2017 Elsevier B.V. All rights reserved.

U bodies respond to nutrient stress in Drosophila

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Buckingham, Mickey; Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk

2011-12-10

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by mutation of the survival motor neuron 1 (SMN1) gene. Cytoplasmic SMN protein-containing granules, known as U snRNP bodies (U bodies), are thought to be responsible for the assembly and storage of small nuclear ribonucleoproteins (snRNPs) which are essential for pre-mRNA splicing. U bodies exhibit close association with cytoplasmic processing bodies (P bodies), which are involved in mRNA decay and translational repression. The close association of the U body and P body in Drosophila resemble that of the stress granule and P body in yeast and mammalian cells. However, it is unknown whether the U body is responsive to any stress. Using Drosophila oogenesis as a model, here we show that U bodies increase in size following nutritional deprivation. Despite nutritional stress, U bodies maintain their close association with P bodies. Our results show that U bodies are responsive to nutrition changes, presumably through the U body-P body pathway.

The Gemin associates of survival motor neuron are required for motor function in Drosophila.

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Borg, Rebecca; Cauchi, Ruben J

2013-01-01

Membership of the survival motor neuron (SMN) complex extends to nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and Unrip. The best-characterised function of this macromolecular machine is the assembly of the Sm-class of uridine-rich small nuclear ribonucleoprotein (snRNP) particles and each SMN complex member has a key role during this process. So far, however, only little is known about the function of the individual Gemin components in vivo. Here, we make use of the Drosophila model organism to uncover loss-of-function phenotypes of Gemin2, Gemin3 and Gemin5, which together with SMN form the minimalistic fly SMN complex. We show that ectopic overexpression of the dead helicase Gem3(ΔN) mutant or knockdown of Gemin3 result in similar motor phenotypes, when restricted to muscle, and in combination cause lethality, hence suggesting that Gem3(ΔN) overexpression mimics a loss-of-function. Based on the localisation pattern of Gem3(ΔN), we predict that the nucleus is the primary site of the antimorphic or dominant-negative mechanism of Gem3(ΔN)-mediated interference. Interestingly, phenotypes induced by human SMN overexpression in Drosophila exhibit similarities to those induced by overexpression of Gem3(ΔN). Through enhanced knockdown we also uncover a requirement of Gemin2, Gemin3 and Gemin5 for viability and motor behaviour, including locomotion as well as flight, in muscle. Notably, in the case of Gemin3 and Gemin5, such function also depends on adequate levels of the respective protein in neurons. Overall, these findings lead us to speculate that absence of any one member is sufficient to arrest the SMN-Gemins complex function in a nucleocentric pathway, which is critical for motor function in vivo.

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

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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.

Control of box C/D snoRNP assembly by N6-methylation of adenine.

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Huang, Lin; Ashraf, Saira; Wang, Jia; Lilley, David Mj

2017-09-01

N 6 -methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N 6 -methyladenine at a key trans Hoogsteen-sugar A·G base pair, of which half are methylated in vivo The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5-kDa protein and the induced folding of the RNA Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N 6 -methylation of adenine prevents the formation of trans Hoogsteen-sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson-Crick base pairs) are more susceptible to disruption by N 6 mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Association of protein C23 with rapidly labeled nucleolar RNA

International Nuclear Information System (INIS)

Herrera, A.H.; Olson, M.O.

1986-01-01

The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [ 3 H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [ 3 H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA

Human neuronal cell protein responses to Nipah virus infection

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Hassan Sharifah

2007-06-01

Full Text Available Abstract Background Nipah virus (NiV, a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches. Results Method for separation of the NiV-infected human neuronal cell proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE was established. At least 800 protein spots were resolved of which seven were unique, six were significantly up-regulated and eight were significantly down-regulated. Six of these altered proteins were identified using mass spectrometry (MS and confirmed using MS/MS. The heterogenous nuclear ribonucleoprotein (hnRNP F, guanine nucleotide binding protein (G protein, voltage-dependent anion channel 2 (VDAC2 and cytochrome bc1 were present in abundance in the NiV-infected SK-N-MC cells in contrast to hnRNPs H and H2 that were significantly down-regulated. Conclusion Several human neuronal cell proteins that are differentially expressed following NiV infection are identified. The proteins are associated with various cellular functions and their abundance reflects their significance in the cytopathologic responses to the infection and the regulation of NiV replication. The potential importance of the ratio of hnRNP F, and hnRNPs H and H2 in regulation of NiV replication, the association of the mitochondrial protein with the cytopathologic responses to the infection and induction of apoptosis are highlighted.

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

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Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Tajima, Shigeru [Department of Virology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640 (Japan); Hikono, Hirokazu; Saito, Takehiko [Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856 (Japan); Aida, Yoko, E-mail: aida@riken.jp [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

2014-07-18

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

International Nuclear Information System (INIS)

Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn; Tajima, Shigeru; Hikono, Hirokazu; Saito, Takehiko; Aida, Yoko

2014-01-01

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC 50 values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets

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

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

2017-04-04

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo's RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subset of Glo's functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

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Joel V. Tamayo

2017-04-01

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

Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition.

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Schlundt, Andreas; Tants, Jan-Niklas; Sattler, Michael

2017-04-15

Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain

Effects of gamma radiation immunogenicity of ribonucleoprotein (RNPs) of rabies virus and purification of anti-RNPs antibodies for diagnosis

International Nuclear Information System (INIS)

Costa, Ana Elena Boamorte da

2010-01-01

The World Health Organization recommends the direct immunofluorescence test for laboratory diagnosis and serological evaluation of rabies. To achieve this test, fluorescent anti-ribo nucleoproteins (RNPs) conjugates, produced from purified IgGs of RNP-immunized animals are employed. The aims of the present study were: investigate the effects of gamma radiation on the immunogenicity of RNPs, as well as to compare two chromatographic methodologies for the purification of anti-RNPs immunoglobulins. Sera from animals immunized with either native or irradiated RNPs were compared by direct immunofluorescence and immuno enzymatic assays. Our results indicate that the animals immunized with irradiated antigen requested a lower number of doses to reach high antibody titers. The immunofluorescence assays indicated that the conjugates produced with the anti-irradiated RNPs IgGs showed similar specificity to its anti-native counterpart, but with a higher definition of the virus inclusions. The purification methods were compared by Bradford and electrophoresis assays. According to the results, we concluded that the affinity-based process resulted in higher yields, lower execution time, and higher purity of the antibodies. (author)

Particle-bound phytochrome: differential pigment release by surfactants, ribonuclease and phospholipase C

International Nuclear Information System (INIS)

Gressel, J.; Quail, P.H.

1976-01-01

Surfactants and hydrolytic enzymes were used to probe the nature of the constituent(s) to which phytochrome binds in particulate fractions from red-irradiated Cucurbita, [ 14 C]-choline and [ 3 H]-uridine pre-labelled tissue was used to monitor the release of phospholipids and RNA by these agents. Ribonuclease (RNase) digestion of 20,000 x g pellets eliminates both the phytochrome and ribonucleprotein (RNP) which cosediment at 31S. Little [ 14 C]-choline occurs in the 31S fraction and the amount is not changed by RNase digestion. This is further evidence that phytochrome binds directly to the RNP in the 31S fraction rather than to any membranous material present. The distribution profile of the RNA in a second (='heavy') phytochrome fraction does not correlate with that of the pigment. This suggests that the phytochrome in this fraction is not bound to RNP. The RNA is of ribosomal origin but much less degraded than that of the 31S RNP and is resistant to RNase digestion. Phospholipase C releases 80% of the [ 14 C]-choline from the 'heavy' fraction without freeing phytochrome. This indicates that the pigment does not bind to the polar head groups of the membrane phospholipids present. Low concentrations of deoxycholate dissociate phytochrome from this fraction without releasing substantial quantities of integral membrane proteins or phospholipids. Some RNP is dislodged by the surfactant but the phytochrome and RNP are not released as a complex. The data suggest that the pigment in the 'heavy' fraction may be loosely bound to a protein constituent rather than to RNP or polar phospholipids. (auth.)

Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport

International Nuclear Information System (INIS)

Kylberg, Karin; Bjoerk, Petra; Fomproix, Nathalie; Ivarsson, Birgitta; Wieslander, Lars; Daneholt, Bertil

2010-01-01

We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.

Environment-dependent regulation of spliceosome activity by the LSM2-8 complex in Arabidopsis.

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Carrasco-López, Cristian; Hernández-Verdeja, Tamara; Perea-Resa, Carlos; Abia, David; Catalá, Rafael; Salinas, Julio

2017-07-07

Spliceosome activity is tightly regulated to ensure adequate splicing in response to internal and external cues. It has been suggested that core components of the spliceosome, such as the snRNPs, would participate in the control of its activity. The experimental indications supporting this proposition, however, remain scarce, and the operating mechanisms poorly understood. Here, we present genetic and molecular evidence demonstrating that the LSM2-8 complex, the protein moiety of the U6 snRNP, regulates the spliceosome activity in Arabidopsis, and that this regulation is controlled by the environmental conditions. Our results show that the complex ensures the efficiency and accuracy of constitutive and alternative splicing of selected pre-mRNAs, depending on the conditions. Moreover, miss-splicing of most targeted pre-mRNAs leads to the generation of nonsense mediated decay signatures, indicating that the LSM2-8 complex also guarantees adequate levels of the corresponding functional transcripts. Interestingly, the selective role of the complex has relevant physiological implications since it is required for adequate plant adaptation to abiotic stresses. These findings unveil an unanticipated function for the LSM2-8 complex that represents a new layer of posttranscriptional regulation in response to external stimuli in eukaryotes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides

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Alessandra Dellavance

2013-06-01

Full Text Available INTRODUCTION: Indirect immunofluorescence on HEp-2 cells is considered the gold standard for the detection of autoantibodies against cellular antigens. However, the culture conditions, cell fixation and permeabilization processes interfere directly in the preservation and spatial distribution of antigens. Therefore, one can assume that certain peculiarities in the processing of cellular substrate may affect the recognition of indirect immunofluorescence patterns associated with several autoantibodies. OBJECTIVE: To evaluate a panel of serum samples representing nuclear, nucleolar, cytoplasmic, mitotic apparatus, and chromosome plate patterns on HEp-2 cell substrates from different suppliers. MATERIALS AND METHODS: Seven blinded observers, independent from the three selected reference centers, evaluated 17 samples yielding different nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns on HEp-2 cell slides from eight different brands. The slides were coded to maintain confidentiality of both brands and participating centers. RESULTS: The 17 HEp-2 cell patterns were identified on most substrates. Nonetheless, some slides showed deficit in the expression of several patterns: nuclear coarse speckled/U1-ribonucleoprotein associated with antibodies against RNP (U1RNP, centromeric protein F (CENP-F, proliferating cell nuclear antigen (PCNA, cytoplasmic fine speckled associated with anti-Jo-1 antibodies (histidyl synthetase, nuclear mitotic apparatus protein 1 (NuMA-1 and nuclear mitotic apparatus protein 2 (NuMA-2. CONCLUSION: Despite the overall good quality of the assessed HEp-2 substrates, there was considerable inconsistency in results among different commercial substrates. The variations may be due to the evaluated batches, hence generalizations cannot be made as to the respective brands. It is recommended that each new batch or new brand be tested with a panel of reference sera representing the various patterns.

Pulmonary hypertension not a major feature of early mixed connective tissue disease: A prospective clinicoserological study

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Haroon N

2005-01-01

Full Text Available Background: Mixed connective tissue disease (MCTD has features common to lupus, scleroderma and myositis with high levels of antibodies to U1 ribonucleoprotein (U1 RNP. Identification of a high incidence of pulmonary artery hypertension (PAH has changed its prospect. We report the largest series from India. Settings and Design: Rheumatology unit of a tertiary care centre in India; prospective. Materials and Methods: Patients seen between January 2002 and June 2004, satisfying the Kasukawa criteria were enrolled. All patients had a complete laboratory work-up including pulmonary function test, 2-D echocardiography, and Schirmer′s test, antinuclear antibodies (ANA and antibodies to extractable nuclear antigens. HRCT of chest was done where indicated. All patients were given standard treatment and followed up regularly. Results: Out of 1500 patients, thirteen (one male were diagnosed to have MCTD. The median follow-up period was 18 months [Interquartile range (IQR 12-22]. The median age of onset of symptoms was 36 years (IQR 22-39 and the median duration of disease was three years (IQR 1.75-4. The most common manifestation was polyarthritis followed by puffy fingers. Sjogren′s syndrome, dysphagia and interstitial lung disease, was present in four, three and two patients respectively. Two patients each had myositis and migraine. None had PAH, serositis or renal involvement. Arthritis, puffy fingers and RaynaudÆs phenomenon were the most common manifestations at onset. All patients were positive for ANA and anti U1 RNP. Two patients each had antibodies to Sm and SSA. Response to treatment also was noted. Conclusion: Pulmonary artery hypertension is not common in early MCTD.

IMP3 RNP Safe Houses Prevent miRNA-Directed HMGA2 mRNA Decay in Cancer and Development

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Lars Jønson

2014-04-01

Full Text Available The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated by let-7, and let-7 antagomiRs make HMGA2 refractory to IMP3. Removal of let-7 target sites eliminates IMP3-dependent stabilization, and IMP3-containing bodies are depleted of Ago1-4 and miRNAs. The relationship between Hmga2 mRNA and IMPs also exists in the developing limb bud, where IMP1-deficient embryos show dose-dependent Hmga2 mRNA downregulation. Finally, IMP3 ribonucleoproteins (RNPs contain other let-7 target mRNAs, including LIN28B, and a global gene set enrichment analysis demonstrates that miRNA-regulated transcripts in general are upregulated following IMP3 induction. We conclude that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression.

RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

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Baoyan Bai

Full Text Available Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA. Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

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

International Nuclear Information System (INIS)

Takagaki, Yoshio; Manley, J.L.; MacDonald, C.C.; Shenk, T.

1992-01-01

Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. The authors 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-cross linked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. They 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 α-helix stabilized by salt bridges is predicted. An ∼270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (∼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

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Indian Academy of Sciences (India)

important roles in different cellular regulatory activities. Recently, a novel RNP complex has been reported in Drosophila. The complex concerns dosage compensation, wherein it promotes the enhancement of transcription of single male X chromosome, so that the quantum of products produced by the single X chromosome ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System.

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Nandal, Anjali; Mallon, Barbara; Telugu, Bhanu P

2017-11-08

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study.

Science.gov (United States)

Chaly, N; Cadrin, M; Kaplan, J G; Brown, D L

1988-01-01

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.

Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

Science.gov (United States)

Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

2015-02-18

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

In vivo effects of traditional Ayurvedic formulations in Drosophila melanogaster model relate with therapeutic applications.

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Vibha Dwivedi

Full Text Available Ayurveda represents the traditional medicine system of India. Since mechanistic details of therapy in terms of current biology are not available in Ayurvedic literature, modern scientific studies are necessary to understand its major concepts and procedures. It is necessary to examine effects of the whole Ayurvedic formulations rather than their "active" components as is done in most current studies.We tested two different categories of formulations, a Rasayana (Amalaki Rasayana or AR, an herbal derivative and a Bhasma (Rasa-Sindoor or RS, an organo-metallic derivative of mercury, for effects on longevity, development, fecundity, stress-tolerance, and heterogeneous nuclear ribonucleoprotein (hnRNP levels of Drosophila melanogaster using at least 200 larvae or flies for each assay.A 0.5% (weight/volume supplement of AR or RS affected life-history and other physiological traits in distinct ways. While the size of salivary glands, hnRNP levels in larval tissues, and thermotolerance of larvae/adult flies improved significantly following feeding either of the two formulations, the median life span and starvation resistance improved only with AR. Feeding on AR or RS supplemented food improved fecundity differently. Feeding of larvae and adults with AR increased the fecundity while the same with RS had opposite effect. On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity. RS feeding did not cause heavy metal toxicity.The present study with two Ayurvedic formulations reveals formulation-specific effects on several parameters of the fly's life, which seem to generally agree with their recommended human usages in Ayurvedic practices. Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

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Clément Monot

2013-05-01

Full Text Available L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP first uses its endonuclease (EN to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A tail, a process known as target-primed reverse transcription (TPRT. Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

Double positive CD4+CD8+ T cells: key suppressive role in the production of autoantibodies in systemic lupus erythematosus

Directory of Open Access Journals (Sweden)

Yongkang Wu

2014-01-01

Full Text Available Background & objectives: The presence of CD4+CD8+ (double positive T cells (DPT in the target organs of several autoimmune diseases has been reported. The aim of this study was to investigate the pathogenic role of DPT in systemic lupus erythematosus (SLE. Methods: A total of 175 SLE cases and 125 matched healthy controls were investigated for CD3+, CD4+, CD8+ lymphocytes and DPT by flow cytometry. Serum samples from SLE patients and controls were tested for antinuclear antibody (ANA, anti-double strain deoxyribonucleic acid (anti-dsDNA, anti-U1 ribonucleoprotein (anti-U1 RNP, anti-sjogren syndrome A (anti-SSA, anti-ribosomal P protein (anti-rib-P, anti-Smith (anti-Sm, anti-Sjogren syndrome B (anti-SSB, complement 3 (C3 and complement 4 (C4. Results: The DPT median and 5-95 per cent range of SLE cases and healthy controls were 0.50 [0.10-2.60] and 0.80 [0.20-2.74] respectively (P<0.001. SLE patients were divided into a ≥1:1000 subgroup and a <1:1000 subgroup according to the ANA titre. The DPT of the former subgroup was significantly lower than that of the latter (P=0.032. The DPT medians of positive subgroups with anti-dsDNA (P<0.001, anti-U1RNP (P=0.018, anti-SSA (P=0.021 or anti-rib-P (P=0.039 were also significantly lower than the negative subgroups. Likewise, DPT was significantly lower in SLE subgroups with low concentration of C3 or C4 than those with high concentration (P<0.006. Interpretation & conclusions: Our findings show that the DPT cells may play a key suppressive role in the production of autoantibodies in SLE. Direct evidence that DPT regulates the pathogenesis of SLE needs to be investigated in future work.

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

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Sudhakaran, Indulekha P; Ramaswami, Mani

2017-05-04

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

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

Science.gov (United States)

Monot, Clément; Kuciak, Monika; Viollet, Sébastien; Mir, Ashfaq Ali; Gabus, Caroline; Darlix, Jean-Luc; Cristofari, Gaël

2013-05-01

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants

DEFF Research Database (Denmark)

Rougemaille, Mathieu; Gudipati, Rajani Kanth; Olesen, Jens Raabjerg

2007-01-01

by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3' 5' exonuclease Rrp6p is required...

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

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Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

2015-08-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA-protein interactions.

Science.gov (United States)

Khanova, Elena; Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

2012-04-01

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

Safe Keeping the Message - mRNP Complexes Tweaking after Transcription

Czech Academy of Sciences Publication Activity Database

Hafidh, Said; Čapková, Věra; Honys, David

2011-01-01

Roč. 722, Jun 2012 (2011), s. 118-136 ISSN 0065-2598 R&D Projects: GA ČR GA522/09/0858; GA MŠk(CZ) LC06004; GA MŠk OC08011; GA MŠk(CZ) OC10054 Institutional research plan: CEZ:AV0Z50380511 Keywords : mature pollen * Arabidopsis Thaliana * messenger RNA localization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.093, year: 2011

FUNCTION OF PHLOEM-BORNE INFORMATION MACROMOLECULES IN INTEGRATING PLANT GROWTH & DEVELOPMENT

Energy Technology Data Exchange (ETDEWEB)

William J. Lucas

2012-11-12

Studies on higher plants have revealed the operation of cell-to-cell and long-distance communication networks that mediate the transport of information macromolecules, such as proteins and RNA. Based on the findings from this DOE-funded project and results from other groups, it is now well established that the enucleate sieve tube system of the angiosperms contains a complex set of proteins including RNA binding proteins as well as a unique population of RNA molecules, comprised of both mRNA and small RNA species. Hetero-grafting experiments demonstrated that delivery of such RNA molecules, into the scion, is highly correlated with changes in developmental phenotypes. Furthermore, over the course of this project, our studies showed that plasmodesmata and the phloem are intimately involved in the local and systemic spread of sequence-specific signals that underlie gene silencing in plants. Major advances were also made in elucidating the underlying mechanisms that operate to mediate the selective entry and exit of proteins and RNA into and out of the phloem translocation stream. Our pioneering studies identified the first plant protein with the capacity to both bind specifically to small RNA molecules (si-RNA) and mediate in the cell-to-cell movement of such siRNA. Importantly, studies conducted with support from this DOE program also yielded a detailed characterization of the first phloem-mobile RNP complex isolated from pumpkin, namely the CmRBP50-RNP complex. This RNP complex was shown to bind, in a sequence-specific manner, to a set of transcripts encoding for transcription factors. The remarkable stability of this CmRBP50-RNP complex allows for long-distance delivery of bound transcripts from mature leaves into developing tissues and organs. Knowledge gained from this project can be used to exert control over the long-distance signaling networks used by plants to integrate their physiological and developmental programs at a whole plant level. Eventually, this

Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors.

Science.gov (United States)

Moriel-Carretero, María; Ovejero, Sara; Gérus-Durand, Marie; Vryzas, Dimos; Constantinou, Angelos

2017-12-04

Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes. © 2017 Moriel-Carretero et al.

miR-132 Regulates Dendritic Spine Structure by Direct Targeting of Matrix Metalloproteinase 9 mRNA.

Science.gov (United States)

Jasińska, Magdalena; Miłek, Jacek; Cymerman, Iwona A; Łęski, Szymon; Kaczmarek, Leszek; Dziembowska, Magdalena

2016-09-01

Mir-132 is a neuronal activity-regulated microRNA that controls the morphology of dendritic spines and neuronal transmission. Similar activities have recently been attributed to matrix metalloproteinase-9 (MMP-9), an extrasynaptic protease. In the present study, we provide evidence that miR-132 directly regulates MMP-9 mRNA in neurons to modulate synaptic plasticity. With the use of luciferase reporter system, we show that miR-132 binds to the 3'UTR of MMP-9 mRNA to regulate its expression in neurons. The overexpression of miR-132 in neurons reduces the level of endogenous MMP-9 protein secretion. In synaptoneurosomes, metabotropic glutamate receptor (mGluR)-induced signaling stimulates the dissociation of miR-132 from polyribosomal fractions and shifts it towards the messenger ribonucleoprotein (mRNP)-containing fraction. Furthermore, we demonstrate that the overexpression of miR-132 in the cultured hippocampal neurons from Fmr1 KO mice that have increased synaptic MMP-9 level provokes enlargement of the dendritic spine heads, a process previously implicated in enhanced synaptic plasticity. We propose that activity-dependent miR-132 regulates structural plasticity of dendritic spines through matrix metalloproteinase 9.

Interactions of a didomain fragment of the Drosophila Sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions

International Nuclear Information System (INIS)

Kim, Insil; Muto, Yutaka; Watanabe, Satoru; Kitamura, Aya; Futamura, Yasuhiro; Yokoyama, Shigeyuki; Hosono, Kazumi; Kawai, Gota; Takaku, Hiroshi; Dohmae, Naoshi; Takio, Koji; Sakamoto, Hiroshi; Shimura, Yoshiro

2000-01-01

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sxl) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5- 2 H]uridine phosphoramidite, and synthesized a series of 2 H-labeled RNAs, in which all of the uridine residues except one were replaced by [5- 2 H]uridine in the target sequence, GU 8 C. By observing the H5-H6 TOCSY cross peaks of the series of 2 H-labeled RNAs complexed with the Sxl RBD1-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU 2 GU 8 , AU 8 , and UAU 8 , were assigned by comparison with those of GU 8 C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex

Interactions of a didomain fragment of the Drosophila Sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions

Energy Technology Data Exchange (ETDEWEB)

Kim, Insil; Muto, Yutaka; Watanabe, Satoru; Kitamura, Aya; Futamura, Yasuhiro; Yokoyama, Shigeyuki [University of Tokyo, Department of Biophysics and Biochemistry, Graduate School of Science (Japan); Hosono, Kazumi; Kawai, Gota; Takaku, Hiroshi [Chiba Institute of Technology, Department of Industrial Chemistry (Japan); Dohmae, Naoshi; Takio, Koji [Institute of Physical and Chemical Research (RIKEN) (Japan); Sakamoto, Hiroshi [Kobe University, Department of Biology, Faculty of Science (Japan); Shimura, Yoshiro [Biomolecular Engineering Research Institute (Japan)

2000-06-15

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sxl) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-{sup 2}H]uridine phosphoramidite, and synthesized a series of {sup 2}H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-{sup 2}H]uridine in the target sequence, GU{sub 8}C. By observing the H5-H6 TOCSY cross peaks of the series of {sup 2}H-labeled RNAs complexed with the Sxl RBD1-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU{sub 2}GU{sub 8}, AU{sub 8}, and UAU{sub 8}, were assigned by comparison with those of GU{sub 8}C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

Science.gov (United States)

Khanova, Elena; Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S.

2012-01-01

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA–protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes. PMID:22332141

Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

NARCIS (Netherlands)

Hennig, Sven; Kong, Geraldine; Mannen, Taro; Sadowska, Agata; Kobelke, Simon; Blythe, Amanda; Knott, Gavin J; Iyer, K Swaminathan; Ho, Diwei; Newcombe, Estella A; Hosoki, Kana; Goshima, Naoki; Kawaguchi, Tetsuya; Hatters, Danny; Trinkle-Mulcahy, Laura; Hirose, Tetsuro; Bond, Charles S; Fox, Archa H

2015-01-01

Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

NARCIS (Netherlands)

Jore, M.M.; Lundgren, N.M.J.; Duijn, van E.; Bultema, J.B.; Westra, E.R.; Oost, van der J.; Brouns, S.J.J.; Beijer, M.R.

2011-01-01

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

NARCIS (Netherlands)

Jore, Matthijs M.; Lundgren, Magnus; van Duijn, Esther; Bultema, Jelle B.; Westra, Edze R.; Waghmare, Sakharam P.; Wiedenheft, Blake; Pul, Uemit; Wurm, Reinhild; Wagner, Rolf; Beijer, Marieke R.; Barendregt, Arjan; Zhou, Kaihong; Snijders, Ambrosius P. L.; Dickman, Mark J.; Doudna, Jennifer A.; Boekema, Egbert J.; Heck, Albert J. R.; van der Oost, John; Brouns, Stan J. J.; Pul, Ümit

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and

RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton.

Science.gov (United States)

Mamon, L A; Ginanova, V R; Kliver, S F; Yakimova, A O; Atsapkina, A A; Golubkova, E V

2017-04-01

The mutual relationship between mRNA and the cytoskeleton can be seen from two points of view. On the one hand, the cytoskeleton is necessary for mRNA trafficking and anchoring to subcellular domains. On the other hand, cytoskeletal growth and rearrangement require the translation of mRNAs that are connected to the cytoskeleton. β-actin mRNA localization may influence dynamic changes in the actin cytoskeleton. In the cytoplasm, long-lived mRNAs exist in the form of RNP (ribonucleoprotein) complexes, where they interact with RNA-binding proteins, including NXF (Nuclear eXport Factor). Dm NXF1 is an evolutionarily conserved protein in Drosophila melanogaster that has orthologs in different animals. The universal function of nxf1 genes is the nuclear export of different mRNAs in various organisms. In this mini-review, we briefly discuss the evidence demonstrating that Dm NXF1 fulfils not only universal but also specialized cytoplasmic functions. This protein is detected not only in the nucleus but also in the cytoplasm. It is a component of neuronal granules. Dm NXF1 marks nuclear division spindles during early embryogenesis and the dense body on one side of the elongated spermatid nuclei. The characteristic features of sbr mutants (sbr 10 and sbr 5 ) are impairment of chromosome segregation and spindle formation anomalies during female meiosis. sbr 12 mutant sterile males with immobile spermatozoa exhibit disturbances in the axoneme, mitochondrial derivatives and cytokinesis. These data allow us to propose that the Dm NXF1 proteins transport certain mRNAs in neurites and interact with localized mRNAs that are necessary for dynamic changes of the cytoskeleton. © 2017 Wiley Periodicals, Inc.

A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.

Science.gov (United States)

Canetta, Elisabetta; Kim, Sang Hyon; Kalinina, Natalia O; Shaw, Jane; Adya, Ashok K; Gillespie, Trudi; Brown, John W S; Taliansky, Michael

2008-02-29

Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

Energy Technology Data Exchange (ETDEWEB)

Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki [National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566 (Japan)

2013-04-30

The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å{sup 3} Da{sup −1} and a solvent content of 59%.

Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

International Nuclear Information System (INIS)

Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

2013-01-01

The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å 3 Da −1 and a solvent content of 59%

Disruption of the murine major vault protein (MVP/LRP) gene does not induce hypersensitivity to cytostatics

NARCIS (Netherlands)

M.H. Mossink (Marieke); A. van Zon (Arend); A.A. Fränzel-Luiten (Erna); M. Schoester (Martijn); V.A. Kickhoefer; G.L. Scheffer (George); R.J. Scheper (Rik); P. Sonneveld (Pieter); E.A.C. Wiemer (Erik)

2002-01-01

textabstractVaults are ribonucleoprotein particles with a distinct structure and a high degree of conservation between species. Although no function has been assigned to the complex yet, there is some evidence for a role of vaults in multidrug resistance. To confirm a direct

Protein composition of catalytically active human telomerase from immortal cells

DEFF Research Database (Denmark)

Cohen, Scott B; Graham, Mark E; Lovrecz, George O

2007-01-01

Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on th...

Efficient Recreation of t(11;22 EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9

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Raul Torres-Ruiz

2017-05-01

Full Text Available Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22 translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1 using end-joining DNA processing factors involved in repair mechanisms, or (2 ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3 all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22 is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22 in human stem cells opens up new avenues in modeling Ewing sarcoma.

The RNA synthesis machinery of negative-stranded RNA viruses

International Nuclear Information System (INIS)

Ortín, Juan; Martín-Benito, Jaime

2015-01-01

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

The RNA synthesis machinery of negative-stranded RNA viruses

Energy Technology Data Exchange (ETDEWEB)

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

2015-05-15

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

Assembly and disassembly of the nucleolus during the cell cycle.

Science.gov (United States)

Hernandez-Verdun, Danièle

2011-01-01

The nucleolus is a large nuclear domain in which transcription, maturation and assembly of ribosomes take place. In higher eukaryotes, nucleolar organization in three sub-domains reflects the compartmentation of the machineries related to active or inactive transcription of the ribosomal DNA, ribosomal RNA processing and assembly with ribosomal proteins of the two (40S and 60S) ribosomal subunits. The assembly of the nucleoli during telophase/early G(1) depends on pre-existing machineries inactivated during prophase (the transcription machinery and RNP processing complexes) and on partially processed 45S rRNAs inherited throughout mitosis. In telophase, the 45S rRNAs nucleate the prenucleolar bodies and order the dynamics of nucleolar assembly. The assembly/disassembly processes of the nucleolus depend on the equilibrium between phosphorylation/dephosphorylation of the transcription machinery and on the RNP processing complexes under the control of the CDK1-cyclin B kinase and PP1 phosphatases. The dynamics of assembly/disassembly of the nucleolus is time and space regulated.

Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation.

Science.gov (United States)

Wang, Peng; Yang, Lijiang; Gao, Yi Qin; Zhao, Xin Sheng

2015-09-03

H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

[Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

Science.gov (United States)

Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

2012-01-01

The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

International Nuclear Information System (INIS)

Peng, Shuxia; Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

2014-01-01

Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance

Human Splicing Finder: an online bioinformatics tool to predict splicing signals.

Science.gov (United States)

Desmet, François-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Béroud, Gwenaëlle; Claustres, Mireille; Béroud, Christophe

2009-05-01

Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-beta Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5' and 3' splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project.

76 FR 40598 - Standard Instrument Approach Procedures, and Takeoff Minimums and Obstacle Departure Procedures...

Science.gov (United States)

2011-07-11

............ Washington Dulles 1/9891 5/26/11 RNAV (RNP) Z RWY 19L, Orig-A. Intl. 28-Jul-11 FL Orlando Orlando Intl....... 1/9893 5/26/11 RNAV (RNP) Z RWY 17L, Orig. 28-Jul-11 FL Orlando Orlando Intl....... 1/9894 5/26/11 RNAV (RNP) Z RWY 36R, Orig. 28-Jul-11 FL Orlando Orlando Intl....... 1/9895 5/26/11 RNAV (RNP) Z RWY...

Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function.

Science.gov (United States)

Cioni, Jean-Michel; Wong, Hovy Ho-Wai; Bressan, Dario; Kodama, Lay; Harris, William A; Holt, Christine E

2018-03-07

The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells

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Liu Chen

2011-09-01

Full Text Available Abstract Interferon Regulatory Factor-3 (IRF-3 plays a central role in the induction of interferon (IFN production and succeeding interferon-stimulated genes (ISG expression en route for restraining hepatitis C virus (HCV infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-β, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR. Peak expression of IFN-α and IFN-β was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M inhibited HCV internal ribosomal entry site (IRES-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.

Evaluation of diagnostic reliability of radionuclide phlebography using 99mTc-MAA to detect deep venous thrombosis; Its role in establishing indications for inferior vena cava filter implantation

International Nuclear Information System (INIS)

Markovic, S.; Odavic, M.; Spaic, R.; Rostovac, M.; Ajdinovic, B.; Matunovic, A.; Raicevic, B.; Prvulov, S.; Kamenica, S.

1991-01-01

Radionuclide phlebography (RNP) of the lower extremities and pelvis was performed using 99m Tc-MAA in 40 patients with pulmonary thromboembolism. Deep venous thrombosis (DVT) was found in all patients, more frequently in the right calf and in the left iliac veins in 20 patients (55%). RNP and contrast phlebography (CP) were performed in 18 patients and confirmed the presence of DVT in all cases. The greatest specificity of RNP was obtained in the left (92.4%) and in the right pelvis (80%). In 12 of 18 patients in whom a cava filter was implanted, specificity of RNP was 100% for the left thigh and 91.7% for other localizations. In establishing indications for cava filter implantation, RNP should be performed prior to CP and bilaterally, but in case of non-indicated CE, RNP findings should be sufficient. (orig.) [de

Cross-Scale Value Trade-Offs in Managing Social-Ecological Systems: The Politics of Scale in Ruaha National Park, Tanzania

Directory of Open Access Journals (Sweden)

Asim Zia

2011-12-01

Full Text Available Management of social-ecological systems takes place amidst complex governance processes and cross-scale institutional arrangements that are mediated through politics of scale. Each management scenario generates distinct cross-scale trade-offs in the distribution of pluralistic values. This study explores the hypothesis that conservation-oriented management scenarios generate higher value for international and national scale social organizations, whereas mixed or more balanced management scenarios generate higher value for local scale social organizations. This hypothesis is explored in the management context of Ruaha National Park (RNP, Tanzania, especially the 2006 expansion of RNP that led to the eviction of many pastoralists and farmers. Five management scenarios for RNP, i.e., national park, game reserve, game control area, multiple use area, and open area, are evaluated in a multicriteria decision analytical framework on six valuation criteria: economic welfare; good governance; socio-cultural values; social equity; ecosystem services; and biodiversity protection; and at three spatial scales: local, national, and international. Based upon this evaluation, we discuss the politics of scale that ensue from the implementation of management alternatives with different mixes of conservation and development goals in social-ecological systems.

Endogenous MCM7 microRNA cluster as a novel platform to multiplex small interfering and nucleolar RNAs for combinational HIV-1 gene therapy.

Science.gov (United States)

Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L; DiGiusto, David L; Rossi, John J

2012-11-01

Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

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

Science.gov (United States)

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

Myosin Va associates with mRNA in ribonucleoprotein particles present in myelinated peripheral axons and in the central nervous system.

Science.gov (United States)

Calliari, Aldo; Farías, Joaquina; Puppo, Agostina; Canclini, Lucía; Mercer, John A; Munroe, David; Sotelo, José R; Sotelo-Silveira, José R

2014-03-01

Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems. Copyright © 2013 Wiley Periodicals, Inc.

Native tandem and ion mobility mass spectrometry highlight structural and modular similarities in clustered-regularly-interspaced shot-palindromic-repeats (CRISPR)-associated protein complexes from Escherichia coli and Pseudomonas aeruginosa.

Science.gov (United States)

van Duijn, Esther; Barbu, Ioana M; Barendregt, Arjan; Jore, Matthijs M; Wiedenheft, Blake; Lundgren, Magnus; Westra, Edze R; Brouns, Stan J J; Doudna, Jennifer A; van der Oost, John; Heck, Albert J R

2012-11-01

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

Directory of Open Access Journals (Sweden)

Marius Rutkauskas

2015-03-01

Full Text Available CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM. Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.

Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9.

Science.gov (United States)

Torres-Ruiz, Raul; Martinez-Lage, Marta; Martin, Maria C; Garcia, Aida; Bueno, Clara; Castaño, Julio; Ramirez, Juan C; Menendez, Pablo; Cigudosa, Juan C; Rodriguez-Perales, Sandra

2017-05-09

Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p.

Science.gov (United States)

Koyama, Masako; Hirano, Hidemi; Shirai, Natsuki; Matsuura, Yoshiyuki

2017-10-01

Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Complete cDNA sequence coding for human docking protein

Energy Technology Data Exchange (ETDEWEB)

Hortsch, M; Labeit, S; Meyer, D I

1988-01-11

Docking protein (DP, or SRP receptor) is a rough endoplasmic reticulum (ER)-associated protein essential for the targeting and translocation of nascent polypeptides across this membrane. It specifically interacts with a cytoplasmic ribonucleoprotein complex, the signal recognition particle (SRP). The nucleotide sequence of cDNA encoding the entire human DP and its deduced amino acid sequence are given.

Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

Science.gov (United States)

Gardano, Laura; Holland, Linda; Oulton, Rena; Le Bihan, Thierry; Harrington, Lea

2012-01-01

Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. PMID:22187156

Aspergillus oryzae AoSO is a novel component of stress granules upon heat stress in filamentous fungi.

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Hsiang-Ting Huang

Full Text Available Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi. The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

Science.gov (United States)

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

76 FR 37263 - Standard Instrument Approach Procedures, and Takeoff Minimums and Obstacle Departure Procedures...

Science.gov (United States)

2011-06-27

..., Amdt 5B 28-Jul-11 SC Allendale Allendale County... 1/2564 6/3/11 GPS RWY 17, Orig-A 28-Jul-11 GA Tifton... RNAV (RNP) Y RWY 9L, Orig-C Hollywood Intl. 28-Jul-11 GA Atlanta Hartsfield-Jackson 1/0500 6/3/11 RNAV (RNP) Z RWY 26R, Orig-A Atlanta Intl. 28-Jul-11 GA Atlanta Hartsfield-Jackson 1/0501 6/3/11 RNAV (RNP...

Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes

Czech Academy of Sciences Publication Activity Database

Dixit, Sameer; Mueller-McNicoll, M.; David, Vojtěch; Zarnack, K.; Ule, J.; Hashimi, Hassan; Lukeš, Julius

2017-01-01

Roč. 8, č. 1 (2017), č. článku e02288-16. ISSN 2150-7511 R&D Projects: GA ČR GA15-21974S Institutional support: RVO:60077344 Keywords : guide RNA * ribonucleoprotein complexes * nucleotide resolution * proteins * DNA * replication * tbrgg2 * subcomplexes * translation Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 6.956, year: 2016

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

International Nuclear Information System (INIS)

Doller, Anke; Badawi, Amel; Schmid, Tobias; Brauß, Thilo; Pleli, Thomas; Meyer zu Heringdorf, Dagmar; Piiper, Albrecht; Pfeilschifter, Josef; Eberhardt, Wolfgang

2015-01-01

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D 1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E 2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on different Hu

Rasputin, more promiscuous than ever: a review of G3BP.

Science.gov (United States)

Irvine, Katharine; Stirling, Renee; Hume, David; Kennedy, Derek

2004-12-01

In this review, we highlight what G3BP's domain structure initially suggested; that G3BPs are "scaffolding" proteins linking signal transduction to RNA metabolism. Whilst it is most attractive to hypothesise about G3BP's role in signalling to mRNA metabolism, it is not known whether all G3BP functions impinge on their RNA-binding activities, so any theories are naturally subject to this qualification. It is hypothesised that, in coordination with an array of other proteins, G3BP, in a phosphorylation-dependent manner, is involved in the post-transcriptional regulation of a subset of mRNAs, at least some of which are in common with those regulated by Hu proteins. These transcripts, partially controlled at the post-transcriptional level by G3BPs, code for proteins important in transcription (e.g. c-Myc) and cytoskeletal arrangement (e.g. Tau), amongst other as yet undetermined pathways. The subtle differences between G3BP family members could dictate binding to a variety of signalling proteins, so each of the G3BPs may participate in different, though possibly related mRNPs, which are assembled in response to different stimuli. The combinatorial nature of the mRNP complex offers a powerful means of regulating gene expression, beyond that provided by a simple mRNA sequence. The ways in which mRNP flexibility and specificity may be harnessed to coordinate gene expression of functionally or structurally related mRNAs are not yet fully appreciated. Characterising mRNP composition and the function/s of mRNP components, such as the G3BPs, will aid in the understanding of how post-transcriptional mechanisms contribute to the global regulation of gene expression.

Structure of the central RNA recognition motif of human TIA-1 at 1.95 A resolution

International Nuclear Information System (INIS)

Kumar, Amit O.; Swenson, Matthew C.; Benning, Matthew M.; Kielkopf, Clara L.

2008-01-01

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95 A resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Directory of Open Access Journals (Sweden)

N.P. Silva

2004-07-01

Full Text Available Cajal bodies (CB are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Association of HLA-DRB1 alleles with susceptibility to mixed connective tissue disease in Polish patients.

Science.gov (United States)

Paradowska-Gorycka, A; Stypińska, B; Olesińska, M; Felis-Giemza, A; Mańczak, M; Czuszynska, Z; Zdrojewski, Z; Wojciechowicz, J; Jurkowska, M

2016-01-01

Mixed connective tissue disease (MCTD) is a systemic autoimmune disease, originally defined as a connective tissue inflammatory syndrome with overlapping features of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM) and systemic sclerosis (SSc), characterized by the presence of antibodies against components of the U1 small nuclear ribonucleoprotein (U1snRNP). The aim of the study was to assess the frequency of (high-resolution-typed) DRB1 alleles in a cohort of Polish patients with MCTD (n = 103). Identification of the variants potentially associated with risk and protection was carried out by comparison with the DKMS Polish Bone Marrow Donor Registry (41306 alleles). DRB1*15:01 (odds ratio (OR): 6.06; 95% confidence interval (CI) 4.55-8.06), DRB1*04 (OR: 3.69; 95% CI 2.69-5.01) and *09:01 (OR: 8.12; 95% CI 2.15-21.75) were identified as risk alleles for MCTD, while HLA-DRB1*07:01 allele was found to be protective (OR: 0.50; 95% CI 0.28-0.83). The carrier frequency of the DRB1*01 was higher in MCTD patients compared with controls, although the differences were not statistically significant. Our results confirm the modulating influence of HLA-DRB1 genotypes on development of connective tissue diseases such as MCTD. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The ErbB family and androgen receptor signaling are targets of Celecoxib in prostate cancer.

Science.gov (United States)

Brizzolara, Antonella; Benelli, Roberto; Venè, Roberta; Barboro, Paola; Poggi, Alessandro; Tosetti, Francesca; Ferrari, Nicoletta

2017-08-01

Inflammation plays a central role in prostate cancer (PCa) development through significant crosstalk between the COX-2-ErbB family receptor network and androgen receptor (AR)-EGFR signaling pathways. The purpose of this work was to determine the ability of the COX-2 inhibitor Celecoxib to modulate the EGFR-AR signaling pathway in androgen-dependent PCa cells and to provide a rationale for its beneficial use in chemopreventive strategies. Functional studies of Celecoxib activity were performed on LNCaP prostate cancer cells. Western blotting, gene expression analysis, dual-luciferase reporter assay and ELISA were applied to assess the Celecoxib mechanisms of action. We found that Celecoxib, through EGF and amphiregulin (AREG) induction, caused EGFR and ErbB2 activation and consequent degradation associated with the inhibition of androgenic signaling. By upregulating the E3 ubiquitin ligase Nrdp1, Celecoxib also efficiently downregulated ErbB3, which is strongly implicated in castration-resistant prostate cancer. Lastly, Celecoxib directly regulated AR transcription and translation independent of ErbB activation by downregulating the RNA binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K). The simultaneous suppression of ErbB kinases and androgen signaling by Celecoxib represents a novel strategy to interrupt the vicious cycle of AR/ErbB cross-talk with the primary purpose of undermining their resilient signaling in prostate cancer progression. Our data provide important premises for the chemopreventive use of Celecoxib in the clinical management of prostate cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

Science.gov (United States)

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

Heterochromatin protein 1 (HP1a positively regulates euchromatic gene expression through RNA transcript association and interaction with hnRNPs in Drosophila.

Directory of Open Access Journals (Sweden)

Lucia Piacentini

2009-10-01

Full Text Available Heterochromatin Protein 1 (HP1a is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases methylate the histone H3 at lysine 9 (H3K9me, creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays, and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms

Adaptation of Pandemic H1N1 Influenza Viruses in Mice▿

Science.gov (United States)

Ilyushina, Natalia A.; Khalenkov, Alexey M.; Seiler, Jon P.; Forrest, Heather L.; Bovin, Nicolai V.; Marjuki, Henju; Barman, Subrata; Webster, Robert G.; Webby, Richard J.

2010-01-01

The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to α2,3 together with decreasing binding to α2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals. PMID:20592084

SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

Directory of Open Access Journals (Sweden)

Xiao-Peng Xiong

2015-08-01

Full Text Available microRNAs (miRNAs are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs that contain Argonaute (AGO family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be

Applying Required Navigation Performance Concept for Traffic Management of Small Unmanned Aircraft Systems

Science.gov (United States)

Jung, Jaewoo; D'Souza, Sarah N.; Johnson, Marcus A.; Ishihara, Abraham K.; Modi, Hemil C.; Nikaido, Ben; Hasseeb, Hashmatullah

2016-01-01

In anticipation of a rapid increase in the number of civil Unmanned Aircraft System(UAS) operations, NASA is researching prototype technologies for a UAS Traffic Management (UTM) system that will investigate airspace integration requirements for enabling safe, efficient low-altitude operations. One aspect a UTM system must consider is the correlation between UAS operations (such as vehicles, operation areas and durations), UAS performance requirements, and the risk to people and property in the operational area. This paper investigates the potential application of the International Civil Aviation Organizations (ICAO) Required Navigation Performance (RNP) concept to relate operational risk with trajectory conformance requirements. The approach is to first define a method to quantify operational risk and then define the RNP level requirement as a function of the operational risk. Greater operational risk corresponds to more accurate RNP level, or smaller tolerable Total System Error (TSE). Data from 19 small UAS flights are used to develop and validate a formula that defines this relationship. An approach to assessing UAS-RNP conformance capability using vehicle modeling and wind field simulation is developed to investigate how this formula may be applied in a future UTM system. The results indicate the modeled vehicles flight path is robust to the simulated wind variation, and it can meet RNP level requirements calculated by the formula. The results also indicate how vehicle-modeling fidelity may be improved to adequately verify assessed RNP level.

Quantitation of antibodies to nucleoribonucleoprotein by ELISA : relation between antibody levels and disease activity in patients with connective tissue disease

NARCIS (Netherlands)

Houtman, PM; Kallenberg, CGM; Limburg, PC; Huitema, MG; van Rijswijk, MH; The, TH

1985-01-01

We describe a solid-phase enzyme-linked immunosorbent assay (ELISA) for quantitation of antibodies to nucleoribonucleoprotein (nRNP/Sm). nRNP/Sm was purified from rabbit thymus acetone powder by immunoaffinity chromatography and characterized by counterimmunoelectrophoresis (CIE) and immunoblotting

77 FR 1013 - Standard Instrument Approach Procedures, and Takeoff Minimums and Obstacle Departure Procedures...

Science.gov (United States)

2012-01-09

... and specifies the types of SIAP and the corresponding effective dates. This amendment also identifies... RNAV (RNP) Z RWY 34R, Orig 9-Feb-12 AZ Prescott Ernest A. Love Field 1/3517 12/21/11 RNAV (RNP) RWY 3R...

Long non-coding RNA discovery across the genus anopheles reveals conserved secondary structures within and beyond the Gambiae complex.

Science.gov (United States)

Jenkins, Adam M; Waterhouse, Robert M; Muskavitch, Marc A T

2015-04-23

Long non-coding RNAs (lncRNAs) have been defined as mRNA-like transcripts longer than 200 nucleotides that lack significant protein-coding potential, and many of them constitute scaffolds for ribonucleoprotein complexes with critical roles in epigenetic regulation. Various lncRNAs have been implicated in the modulation of chromatin structure, transcriptional and post-transcriptional gene regulation, and regulation of genomic stability in mammals, Caenorhabditis elegans, and Drosophila melanogaster. The purpose of this study is to identify the lncRNA landscape in the malaria vector An. gambiae and assess the evolutionary conservation of lncRNAs and their secondary structures across the Anopheles genus. Using deep RNA sequencing of multiple Anopheles gambiae life stages, we have identified 2,949 lncRNAs and more than 300 previously unannotated putative protein-coding genes. The lncRNAs exhibit differential expression profiles across life stages and adult genders. We find that across the genus Anopheles, lncRNAs display much lower sequence conservation than protein-coding genes. Additionally, we find that lncRNA secondary structure is highly conserved within the Gambiae complex, but diverges rapidly across the rest of the genus Anopheles. This study offers one of the first lncRNA secondary structure analyses in vector insects. Our description of lncRNAs in An. gambiae offers the most comprehensive genome-wide insights to date into lncRNAs in this vector mosquito, and defines a set of potential targets for the development of vector-based interventions that may further curb the human malaria burden in disease-endemic countries.

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Science.gov (United States)

Schmidt, Carla; Grønborg, Mads; Deckert, Jochen; Bessonov, Sergey; Conrad, Thomas; Lührmann, Reinhard; Urlaub, Henning

2014-01-01

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique. PMID:24448447

Sequence Classification: 893627 [

Lifescience Database Archive (English)

Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|6325043|ref|NP_015111.1| Component of U2 snRNP; disrupti...on causes reduced U2 snRNP levels; physically interacts with Msl1p; putative homolo

Telomere Shortening in Neurological Disorders: An Abundance of Unanswered Questions

OpenAIRE

Eitan, Erez; Hutchison, Emmette R.; Mattson, Mark P.

2014-01-01

Telomeres, ribonucleoprotein complexes that cap eukaryotic chromosomes, typically shorten in leukocytes with aging. Aging is a primary risk factor for neurodegenerative disease (ND), and a common assumption has arisen that leukocyte telomere length (LTL) can serve as a predictor of neurological disease. However, the evidence for shorter LTL in Alzheimer’s and Parkinson’s patients is inconsistent. The diverse causes of telomere shortening may explain variability in LTL between studies and indi...

Role of Nab2 in RNA metabolism in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Olszewski, Pawel

RNP assembly generates errors, which are tracked down by nuclear surveillance mechanisms. Thus, not surprisingly, protein components of mRNPs have multiple functions in the biogenesis of the particle but also interact with the surveillance machinery. An example of such a multifunctional factor is the S......RNP components showed changes in the Nab2 interactome in the absence of Rrp6, and also suggested that Nab2 binds to multiple sites on the mRNA. Wide-range proteomic analysis of Nab2 immunoprecipitates revealed association with C/D box snoRNP components. These novel interactions were supported by the presence...

Experimental implementation and proof of principle for a radionuclidic purity test solely based on half-life measurement

DEFF Research Database (Denmark)

Jørgensen, Thomas; Jensen, Mikael

2016-01-01

have developed an GUI application for use as an easy and automated test tool in the production procedure.The test results show that this method fully complies with the requirements in the European Pharmacopoeia (Eur. Ph.) for RNP of FDG and F-18 Sodium Fluoride. (C) 2015 Elsevier Ltd. All rights......In this paper we present the results of an experimental implementation of the method (Jorgensen et al., 2012) for testing the radionuclidic purity (RNP) of F-18 compounds.The overall limitations of the experimental methods and their possible impacts on RNP detectability have been identified. We...

4D Dynamic Required Navigation Performance Final Report

Science.gov (United States)

Finkelsztein, Daniel M.; Sturdy, James L.; Alaverdi, Omeed; Hochwarth, Joachim K.

2011-01-01

New advanced four dimensional trajectory (4DT) procedures under consideration for the Next Generation Air Transportation System (NextGen) require an aircraft to precisely navigate relative to a moving reference such as another aircraft. Examples are Self-Separation for enroute operations and Interval Management for in-trail and merging operations. The current construct of Required Navigation Performance (RNP), defined for fixed-reference-frame navigation, is not sufficiently specified to be applicable to defining performance levels of such air-to-air procedures. An extension of RNP to air-to-air navigation would enable these advanced procedures to be implemented with a specified level of performance. The objective of this research effort was to propose new 4D Dynamic RNP constructs that account for the dynamic spatial and temporal nature of Interval Management and Self-Separation, develop mathematical models of the Dynamic RNP constructs, "Required Self-Separation Performance" and "Required Interval Management Performance," and to analyze the performance characteristics of these air-to-air procedures using the newly developed models. This final report summarizes the activities led by Raytheon, in collaboration with GE Aviation and SAIC, and presents the results from this research effort to expand the RNP concept to a dynamic 4D frame of reference.

Improved methods to determine radionuclidic purity of F-18 compounds

DEFF Research Database (Denmark)

Jørgensen, Thomas; Micheelsen, Mille Ankerstjerne; Jensen, Mikael

2012-01-01

Current revisions of monographs for F-18 pharmaceuticals in the European Pharmacopoeia (Ph. Eur.) (Ph. Eur., 2011) call for a radionuclidic purity (RNP) of or better than 99.9%. However, the current method is not sufficient nor effective for testing this required RNP level. We present a theoretical...

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

Energy Technology Data Exchange (ETDEWEB)

Doller, Anke; Badawi, Amel [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Schmid, Tobias; Brauß, Thilo [Institut für Biochemie I (Pathobiochemie), Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pleli, Thomas [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Meyer zu Heringdorf, Dagmar [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Piiper, Albrecht [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pfeilschifter, Josef [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Eberhardt, Wolfgang, E-mail: w.eberhardt@em.uni-frankfurt.de [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany)

2015-01-01

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on

Concerted effects of heterogeneous nuclear ribonucleoprotein C1/C2 to control vitamin D-directed gene transcription and RNA splicing in human bone cells.

Science.gov (United States)

Zhou, Rui; Park, Juw Won; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Zavala, Kathryn; Sea, Jessica L; Lu, Zhi-Xiang; Xu, Jianzhong; Adams, John S; Xing, Yi; Hewison, Martin

2017-01-25

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH) 2 D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH) 2 D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH) 2 D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH) 2 D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH) 2 D-mediated induction of transcription. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

OpenAIRE

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

2003-01-01

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

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

Directory of Open Access Journals (Sweden)

Dominik M Meinel

2013-11-01

Full Text Available Messenger RNA (mRNA synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5 diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1 phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

Science.gov (United States)

Meinel, Dominik M; Burkert-Kautzsch, Cornelia; Kieser, Anja; O'Duibhir, Eoghan; Siebert, Matthias; Mayer, Andreas; Cramer, Patrick; Söding, Johannes; Holstege, Frank C P; Sträßer, Katja

2013-11-01

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

Science.gov (United States)

Fu, Xiang-Dong

2017-04-01

EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines

Science.gov (United States)

Tosar, Juan Pablo; Gámbaro, Fabiana; Sanguinetti, Julia; Bonilla, Braulio; Witwer, Kenneth W.; Cayota, Alfonso

2015-01-01

Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19–60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5′ tRNA halves and 5′ RNA Y4-derived fragments of 31–33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space. PMID:25940616

Dynamic Changes in the Protein Localization in the Nuclear Environment in Pancreatic β-Cell after Brief Glucose Stimulation

DEFF Research Database (Denmark)

Kang, Taewook; Jensen, Pia; Solovyeva, Vita

2018-01-01

, we identified 20 components of the nuclear organization processes, including nuclear pore organization, ribonucleoprotein complex, and pre-mRNA transcription. We found alteration of the nuclear pore complex, together with calcium/calmodulin-binding chaperones that facilitate protein and RNA import......Characterization of molecular mechanisms underlying pancreatic β-cell function in relation to glucose-stimulated insulin secretion is incomplete, especially with respect to global response in the nuclear environment. We focus on the characterization of proteins in the nuclear environment of β...... the nucleus and the cytoplasm is an important process, highly involved in the initial molecular mechanism underlying glucose-stimulated insulin secretion in pancreatic β-cells....

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

In an attempt to characterize the interaction occurring between cellular proteins and hnRNP L, yeast two-hybrid screening was conducted using a HeLa cDNA library. Some of the cDNA clones were found to harbour a partial human hnRNP D/AUF1 cDNA (GeneBank accession number NM_031369). In this study, we ...

Effects of gamma radiation immunogenicity of ribonucleoprotein (RNPs) of rabies virus and purification of anti-RNPs antibodies for diagnosis; Efeitos da radiacao gama na imunogenicidade das ribonucleoproteinas (RNPs) do virus da raiva e purificacao de anticorpos anti-RNPs para diagnostico

Energy Technology Data Exchange (ETDEWEB)

Costa, Ana Elena Boamorte da

2010-07-01

The World Health Organization recommends the direct immunofluorescence test for laboratory diagnosis and serological evaluation of rabies. To achieve this test, fluorescent anti-ribo nucleoproteins (RNPs) conjugates, produced from purified IgGs of RNP-immunized animals are employed. The aims of the present study were: investigate the effects of gamma radiation on the immunogenicity of RNPs, as well as to compare two chromatographic methodologies for the purification of anti-RNPs immunoglobulins. Sera from animals immunized with either native or irradiated RNPs were compared by direct immunofluorescence and immuno enzymatic assays. Our results indicate that the animals immunized with irradiated antigen requested a lower number of doses to reach high antibody titers. The immunofluorescence assays indicated that the conjugates produced with the anti-irradiated RNPs IgGs showed similar specificity to its anti-native counterpart, but with a higher definition of the virus inclusions. The purification methods were compared by Bradford and electrophoresis assays. According to the results, we concluded that the affinity-based process resulted in higher yields, lower execution time, and higher purity of the antibodies. (author)

A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

International Nuclear Information System (INIS)

Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E.

2005-01-01

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with β-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins

Methylation of nucleolar RNA in HeLa cells studied by autoradiography

International Nuclear Information System (INIS)

Cervera, J.; Martinez, A.; Renau-Piqueras, J.

1984-01-01

Methylation of nucleolar RNA was studied by autoradiography in HeLa cells using L-[methyl- 3 H]methionine and S-adenosyl-L-[methyl- 3 H]methionine as radioactive precursors. Pulse-labeling experiments show that nucleolar RNA methylation occurs on the newly synthesized RNA at the nucleolar fibrillar RNP component and mostly on the fibrillar ring of fibrillar centers, where pre-rRNA is being synthesized. Pulse-chase experiments show a shift of silver grains from the nucleolar fibrillar RNP component to the nucleolar granular component first and then to the cytoplasm. Labeling of nucleolar RNA via specific methylation permits the study of intranucleolar processing of pre-rRNA and confirms the sequence of labeling of the two nucleolar RNP components observed with radioactive uridine

Control of flowering and cell fate by LIF2, an RNA binding partner of the polycomb complex component LHP1.

Directory of Open Access Journals (Sweden)

David Latrasse

Full Text Available Polycomb Repressive Complexes (PRC modulate the epigenetic status of key cell fate and developmental regulators in eukaryotes. The chromo domain protein like heterochromatin protein1 (LHP1 is a subunit of a plant PRC1-like complex in Arabidopsis thaliana and recognizes histone H3 lysine 27 trimethylation, a silencing epigenetic mark deposited by the PRC2 complex. We have identified and studied an LHP1-Interacting Factor2 (LIF2. LIF2 protein has RNA recognition motifs and belongs to the large hnRNP protein family, which is involved in RNA processing. LIF2 interacts in vivo, in the cell nucleus, with the LHP1 chromo shadow domain. Expression of LIF2 was detected predominantly in vascular and meristematic tissues. Loss-of-function of LIF2 modifies flowering time, floral developmental homeostasis and gynoecium growth determination. lif2 ovaries have indeterminate growth and produce ectopic inflorescences with severely affected flowers showing proliferation of ectopic stigmatic papillae and ovules in short-day conditions. To look at how LIF2 acts relative to LHP1, we conducted transcriptome analyses in lif2 and lhp1 and identified a common set of deregulated genes, which showed significant enrichment in stress-response genes. By comparing expression of LHP1 targets in lif2, lhp1 and lif2 lhp1 mutants we showed that LIF2 can either antagonize or act with LHP1. Interestingly, repression of the FLC floral transcriptional regulator in lif2 mutant is accompanied by an increase in H3K27 trimethylation at the locus, without any change in LHP1 binding, suggesting that LHP1 is targeted independently from LIF2 and that LHP1 binding does not strictly correlate with gene expression. LIF2, involved in cell identity and cell fate decision, may modulate the activity of LHP1 at specific loci, during specific developmental windows or in response to environmental cues that control cell fate determination. These results highlight a novel link between plant RNA

Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation

DEFF Research Database (Denmark)

Libri, Domenico; Dower, Ken; Boulay, Jocelyne

2002-01-01

Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional el...... results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p....

Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

Directory of Open Access Journals (Sweden)

Youn-Bok Lee

2013-12-01

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

Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P2.

Science.gov (United States)

Kakisaka, Michinori; Yamada, Kazunori; Yamaji-Hasegawa, Akiko; Kobayashi, Toshihide; Aida, Yoko

2016-09-01

To be incorporated into progeny virions, the viral genome must be transported to the inner leaflet of the plasma membrane (PM) and accumulate there. Some viruses utilize lipid components to assemble at the PM. For example, simian virus 40 (SV40) targets the ganglioside GM1 and human immunodeficiency virus type 1 (HIV-1) utilizes phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. Recent studies clearly indicate that Rab11-mediated recycling endosomes are required for influenza A virus (IAV) trafficking of vRNPs to the PM but it remains unclear how IAV vRNP localized or accumulate underneath the PM for viral genome incorporation into progeny virions. In this study, we found that the second intrinsically disordered region (IDR2) of NP regulates two binding steps involved in viral genome packaging. First, IDR2 facilitates NP oligomer binding to viral RNA to form vRNP. Secondly, vRNP assemble by interacting with PI(4,5)P2 at the PM via IDR2. These findings suggest that PI(4,5)P2 functions as the determinant of vRNP accumulation at the PM. Copyright © 2016 Elsevier Inc. All rights reserved.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

Science.gov (United States)

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-05-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling.

Science.gov (United States)

Cajigas, Ivelisse; Leib, David E; Cochrane, Jesse; Luo, Hao; Swyter, Kelsey R; Chen, Sean; Clark, Brian S; Thompson, James; Yates, John R; Kingston, Robert E; Kohtz, Jhumku D

2015-08-01

Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNF-related chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNA-dependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNA-mediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin-Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNA-binding and DLX1-binding domains. © 2015. Published by The Company of Biologists Ltd.

pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion.

Science.gov (United States)

Batishchev, O V; Shilova, L A; Kachala, M V; Tashkin, V Y; Sokolov, V S; Fedorova, N V; Baratova, L A; Knyazev, D G; Zimmerberg, J; Chizmadzhev, Y A

2016-01-01

Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ~6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ~5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In

Introducing a Novel Hybrid Artificial Intelligence Algorithm to Optimize Network of Industrial Applications in Modern Manufacturing

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Aydin Azizi

2017-01-01

Full Text Available Recent advances in modern manufacturing industries have created a great need to track and identify objects and parts by obtaining real-time information. One of the main technologies which has been utilized for this need is the Radio Frequency Identification (RFID system. As a result of adopting this technology to the manufacturing industry environment, RFID Network Planning (RNP has become a challenge. Mainly RNP deals with calculating the number and position of antennas which should be deployed in the RFID network to achieve full coverage of the tags that need to be read. The ultimate goal of this paper is to present and evaluate a way of modelling and optimizing nonlinear RNP problems utilizing artificial intelligence (AI techniques. This effort has led the author to propose a novel AI algorithm, which has been named “hybrid AI optimization technique,” to perform optimization of RNP as a hard learning problem. The proposed algorithm is composed of two different optimization algorithms: Redundant Antenna Elimination (RAE and Ring Probabilistic Logic Neural Networks (RPLNN. The proposed hybrid paradigm has been explored using a flexible manufacturing system (FMS, and results have been compared with Genetic Algorithm (GA that demonstrates the feasibility of the proposed architecture successfully.

Making RISC.

Science.gov (United States)

Kawamata, Tomoko; Tomari, Yukihide

2010-07-01

It is well established that 20- to 30-nt small RNAs, including small interfering RNAs, microRNAs and Piwi-interacting RNAs, play crucial roles in regulating gene expression and control a surprisingly diverse array of biological processes. These small RNAs cannot work alone: they must form effector ribonucleoprotein complexes - RNA-induced silencing complexes (RISCs) - to exert their function. Thus, RISC assembly is a key process in small RNA-mediated silencing. Recent biochemical analyses of RISC assembly, together with new structural studies of Argonaute, the core protein component of RISC, suggest a revised view of how mature RISC, which contains single-stranded guide RNA, is built from small RNAs that are born double-stranded. Copyright 2010 Elsevier Ltd. All rights reserved.

Oral nanotherapeutics: Redox nanoparticles attenuate ultraviolet B radiation-induced skin inflammatory disorders in Kud:Hr- hairless mice.

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Feliciano, Chitho P; Nagasaki, Yukio

2017-10-01

The active participation of an anti-inflammatory drug in the biological pathways of inflammation is crucial for the achievement of beneficial and therapeutic effects. This study demonstrated the development of redox nanoparticles that can circulate in the blood at significantly high levels, thus increasing their efficacy as an oral treatment against the deleterious effects of reactive oxygen species (ROS) in an in vivo inflammatory skin model. To confirm the blood bioavailability of the nanoparticles, mice were injected with the nanoparticles solution (RNP N ) via oral gavage. Using electron spin resonance and radioactive labeling techniques, the blood circulation of the redox polymer that forms the nanoparticles was confirmed 24 h after oral administration. This contrasted with its low molecular weight counterpart (NH 2 -TEMPO), which peaked 15 min post injection and was found to be cleared rapidly within minutes after the peak. We then tested its efficacy in the inflammatory skin model. Kud:Hr-hairless mice were irradiated with UVB (302 nm) to induce skin damage and inflammation. Throughout the entire period of UVB irradiation, RNP N was administered to mice by free drinking. NH 2 -TEMPO was used as the control. The results showed that oral supplementation of RNP N significantly improved the therapeutic effects of the core nitroxide radical compared with its low molecular weight counterpart. Furthermore, RNP N significantly reduced UVB-induced skin aging, epidermal thickening, edema, erythema, skin lesions, and various pathological skin inflammatory disorders in vivo. From the obtained data, we concluded that the use of long-circulating redox nanoparticles (RNP N ) provided an effective treatment against the damaging effects of excessive ROS in the body. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential association of protein subunits with the human RNase MRP and RNase P complexes.

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Welting, Tim J M; Kikkert, Bastiaan J; van Venrooij, Walther J; Pruijn, Ger J M

2006-07-01

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.

KPNB1 mediates PER/CRY nuclear translocation and circadian clock function.

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Lee, Yool; Jang, A Reum; Francey, Lauren J; Sehgal, Amita; Hogenesch, John B

2015-08-29

Regulated nuclear translocation of the PER/CRY repressor complex is critical for negative feedback regulation of the circadian clock of mammals. However, the precise molecular mechanism is not fully understood. Here, we report that KPNB1, an importin β component of the ncRNA repressor of nuclear factor of activated T cells (NRON) ribonucleoprotein complex, mediates nuclear translocation and repressor function of the PER/CRY complex. RNAi depletion of KPNB1 traps the PER/CRY complex in the cytoplasm by blocking nuclear entry of PER proteins in human cells. KPNB1 interacts mainly with PER proteins and directs PER/CRY nuclear transport in a circadian fashion. Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner. Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies. Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

Population Viability Analysis of the Endangered Roan Antelope in Ruma National Park, Kenya, and Implications for Management.

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Kimanzi, Johnstone K

2018-01-01

Population viability analysis (PVA) was used to (1) establish causes of roan population decline for the past 30 years in Ruma National Park (RNP), the only park where wild roans remain in Kenya, and (2) predict the probability of roan persistence under existing and alternative management options. PVA was done using long-term data based on population dynamics, life history, climatic conditions, and expert knowledge. Poaching was identified as the main cause of roan decline in RNP. Several antipoaching and prioritized habitat management interventions to promote population recovery and sustainable conservation of roans are described. PVA predictions indicated that, without these interventions, the roan population cannot persist more than 3 decades. Furthermore, ensuring sustainable conservation of roans in RNP will boost tourism in Western Kenyan and thus alleviate poverty in this part of the country. Improved income from tourism will reduce the possible pressures from hunting and give greater incentives for local people to be actively engaged in roan conservation.

Efficacy and Safety of Medicinal Plants or Related Natural Products for Fibromyalgia: A Systematic Review

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Simone de Souza Nascimento

2013-01-01

Full Text Available To assess the effects of medicinal plants (MPs or related natural products (RNPs on fibromyalgia (FM patients, we evaluate the possible benefits and advantages of MP or RNP for the treatment of FM based on eight randomized placebo-controlled trials (RCTs involving 475 patients. The methodological quality of all studies included was determined according to JADAD and “Risk of Bias” with the criteria in the Cochrane Handbook for Systematic Reviews of Interventions 5.1.0. Evidence suggests significant benefits of MP or RNP in sleep disruption, pain, depression, joint stiffness, anxiety, physical function, and quality of life. Our results demonstrated that MP or RNP had significant effects on improving the symptoms of FM compared to conventional drug or placebo; longer tests are required to determine the duration of the treatment and characterize the long-term safety of using MP, thus suggesting effective alternative therapies in the treatment of pain with minimized side effects.

Hsp90 inhibitors reduce influenza virus replication in cell culture

International Nuclear Information System (INIS)

Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin; Brownlee, George

2008-01-01

The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses

Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

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Toro Nicolás

2011-05-01

Full Text Available Abstract Background Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ' in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron. Results In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo. Conclusions The excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.

Role of a redox-based methylation switch in mRNA life cycle ( pre- & post- transcriptional maturation and protein turnover : Implications in neurological disorders

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MALAV SUCHIN TRIVEDI

2012-06-01

Full Text Available Homeostatic synaptic scaling in response to neuronal stimulus or activation, as well as due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions. Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic. This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition and behavior. Thus a regulatory switch, controlling the lifespan, maturation and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at 1.The pre-transcription level, by regulating precursor-RNA (pre-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and 2. the post-transcription level by modulating the regulatory functions of ribonucleoproteins (RNP and RNA binding proteins (RNABP in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione antioxidant levels, the redox status of neurons might be the central regulatory switch for methylation

La marque «Parc Naturel Régional» :un dispositif original pour valoriser des stratégies de développement durable fondées sur les ressources agricoles et agroalimentaires des territoires insulaires ultramarins

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Armelle Caron

2009-06-01

Full Text Available Les Parcs Naturels Régionaux (PNR français créés en 1967 constituent de véritables laboratoires pour la mise en œuvre d’un développement durable avant la lettre. Ils ont expérimenté diverses initiatives visant à valoriser la conservation des patrimoines naturel et culturel de leur territoire, notamment une politique de marquage de produits et de services territorialement ancrés. Dans cet article, nous nous interrogeons sur la pertinence de ces instruments de développement local pour les territoires insulaires à partir de l'exemple de la politique de marquage mise en œuvre par le PNR de la Martinique. Nous intéressant plus particulièrement aux impacts des démarches de marquage en termes de dynamique d’action collective, nous centrons notre analyse sur la dimension « sociale » de la durabilité.French Regional Nature Parks (RNP, created in 1967, can be considered as precursory sustainable development laboratories. Different initiatives dedicated to promote local development and draw benefits from the improved status of sustainably managed areas have been implemented in the RNP and among them a labelling policy through the development of RNP collective trademarks. In this article, we wonder about the consistency of this RNP labelling policy for the French overseas department of Martinique. According to our analytical references (local governance and collective action, we focus on the social dimension of sustainable development.

Evaluation of the Terminal Sequencing and Spacing System for Performance Based Navigation Arrivals

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Thipphavong, Jane; Jung, Jaewoo; Swenson, Harry N.; Martin, Lynne; Lin, Melody; Nguyen, Jimmy

2013-01-01

NASA has developed the Terminal Sequencing and Spacing (TSS) system, a suite of advanced arrival management technologies combining timebased scheduling and controller precision spacing tools. TSS is a ground-based controller automation tool that facilitates sequencing and merging arrivals that have both current standard ATC routes and terminal Performance-Based Navigation (PBN) routes, especially during highly congested demand periods. In collaboration with the FAA and MITRE's Center for Advanced Aviation System Development (CAASD), TSS system performance was evaluated in human-in-the-loop (HITL) simulations with currently active controllers as participants. Traffic scenarios had mixed Area Navigation (RNAV) and Required Navigation Performance (RNP) equipage, where the more advanced RNP-equipped aircraft had preferential treatment with a shorter approach option. Simulation results indicate the TSS system achieved benefits by enabling PBN, while maintaining high throughput rates-10% above baseline demand levels. Flight path predictability improved, where path deviation was reduced by 2 NM on average and variance in the downwind leg length was 75% less. Arrivals flew more fuel-efficient descents for longer, spending an average of 39 seconds less in step-down level altitude segments. Self-reported controller workload was reduced, with statistically significant differences at the p less than 0.01 level. The RNP-equipped arrivals were also able to more frequently capitalize on the benefits of being "Best-Equipped, Best- Served" (BEBS), where less vectoring was needed and nearly all RNP approaches were conducted without interruption.

Autoantibodies in scleroderma and their association with the clinical profile of the disease. A study of 66 patients from southern Brazil.

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Skare, Thelma Larocca; Fonseca, Adriano Erlon; Luciano, Alan Campos; Azevedo, Pedro Ming

2011-01-01

Scleroderma is a fairly rare connective tissue disease whose autoantibody profile is associated with different clinical manifestations. The prevalence of autoantibodies in scleroderma is influenced by race and genetics. To study the prevalence of anti-Scl-70, anti-centromere (ACA) and anti-U1-RNP antibodies in patients with scleroderma in southern Brazil and verify their association with clinical manifestations of the disease. A retrospective study involving 66 patients with scleroderma for the presence of anti-Scl-70, anti-centromere and anti-U1-RNP and of clinical manifestations such as Raynaud's phenomenon, digital micro scars, digital necrosis, telangiectasias, calcinosis, pulmonary fibrosis, pleuritis, pericarditis, cardiomyopathy, arthralgia and arthritis, skin sclerosis, joint contractures, tendon friction rubs, pulmonary hypertension, esophageal disorders and renal crisis. The prevalence of anti-Scl-70 was 17.8% , that of ACA was 33.3% and the prevalence of U1 RNP was 11.8%. Anti-Scl-70 was associated with the diffuse form of the disease (p = 0.015), presence of cardiomyopathies (p = 0.016) and digital micro scars (p = 0.05). Anti-centromere was more common in the limited form, although it was not statistically significant, and had a protective role associated with cardiomyopathies (p = 0.005). Anti-U1-RNP was more common in the overlap forms (p = 0.0004). The prevalence and profile of clinical associations of autoantibodies in Brazilian patients with scleroderma are similar to those found in the literature.

Autoanticorpos em esclerodermia e sua associação ao perfil clínico da doença: estudo em 66 pacientes do sul do Brasil Autoantibodies in scleroderma and their association with the clinical profile of the disease: a study of 66 patients from southern Brazil

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Thelma Larocca Skare

2011-12-01

Full Text Available FUNDAMENTOS: A esclerodermia é uma colagenose relativamente rara, cujo perfil de autoanticorpos está associado a diferentes manifestações clínicas. A prevalência de autoanticorpos na esclerodermia sofre influência racial e genética. OBJETIVO: Estudar a prevalência dos anticorpos anti-Scl-70, anticentrômero e anti-U1-RNP em pacientes com esclerodermia do sul do Brasil e verificar suas associações às manifestações clínicas. MÉTODOS: Estudo retrospectivo de análise de 66 pacientes com esclerodermia para presença de anti-Scl-70, anticentrômero (ACA e anti-U1-RNP e de manifestações clínicas como: Raynaud, cicatrizes estelares, necrose digital, telangiectasias, calcinose, fibrose pulmonar, pleurites, pericardites, miocardiopatias, artralgias e artrites, grau de esclerose da pele, contraturas articulares e atritos de tendão, hipertensão pulmonar, manifestações esofágicas e crise renal. RESULTADOS: A prevalência do anti-Scl-70 foi de 17,8%, a do ACA, de 33,3%, e a do U1 RNP foi de 11,8 %. O anti-Scl-70 estava associado à forma difusa da doença (p=0,015, presença de miocardiopatias (p=0,016 e de cicatrizes estelares (p=0,05; o anticentrômero foi mais comum na forma limitada, embora sem significância estatística e mostrou-se protetor para as miocardiopatias (p=0,005. O anti-U1-RNP foi mais comum nas formas de superposição (p=0,0004. CONCLUSÃO: A prevalência e o perfil de associações clínicas dos autoanticorpos em esclerodermia de pacientes brasileiros assemelham-se aos da literatura mundial.BACKGROUND: Scleroderma is a fairly rare connective tissue disease whose autoantibody profile is associated with different clinical manifestations. The prevalence of autoantibodies in scleroderma is influenced by race and genetics. OBJECTIVE: To study the prevalence of anti-Scl-70, anti-centromere (ACA and anti-U1-RNP antibodies in patients with scleroderma in southern Brazil and verify their association with clinical

Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

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Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

2007-07-12

Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17A (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Communication complexity and information complexity

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Pankratov, Denis

Information complexity enables the use of information-theoretic tools in communication complexity theory. Prior to the results presented in this thesis, information complexity was mainly used for proving lower bounds and direct-sum theorems in the setting of communication complexity. We present three results that demonstrate new connections between information complexity and communication complexity. In the first contribution we thoroughly study the information complexity of the smallest nontrivial two-party function: the AND function. While computing the communication complexity of AND is trivial, computing its exact information complexity presents a major technical challenge. In overcoming this challenge, we reveal that information complexity gives rise to rich geometrical structures. Our analysis of information complexity relies on new analytic techniques and new characterizations of communication protocols. We also uncover a connection of information complexity to the theory of elliptic partial differential equations. Once we compute the exact information complexity of AND, we can compute exact communication complexity of several related functions on n-bit inputs with some additional technical work. Previous combinatorial and algebraic techniques could only prove bounds of the form theta( n). Interestingly, this level of precision is typical in the area of information theory, so our result demonstrates that this meta-property of precise bounds carries over to information complexity and in certain cases even to communication complexity. Our result does not only strengthen the lower bound on communication complexity of disjointness by making it more exact, but it also shows that information complexity provides the exact upper bound on communication complexity. In fact, this result is more general and applies to a whole class of communication problems. In the second contribution, we use self-reduction methods to prove strong lower bounds on the information

Structural analysis of the complex between influenza B nucleoprotein and human importin-α.

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Labaronne, Alice; Milles, Sigrid; Donchet, Amélie; Jensen, Malene Ringkjøbing; Blackledge, Martin; Bourhis, Jean-Marie; Ruigrok, Rob W H; Crépin, Thibaut

2017-12-07

Influenza viruses are negative strand RNA viruses that replicate in the nucleus of the cell. The viral nucleoprotein (NP) is the major component of the viral ribonucleoprotein. In this paper we show that the NP of influenza B has a long N-terminal tail of 70 residues with intrinsic flexibility. This tail contains the Nuclear Location Signal (NLS). The nuclear trafficking of the viral components mobilizes cellular import factors at different stages, making these host-pathogen interactions promising targets for new therapeutics. NP is imported into the nucleus by the importin-α/β pathway, through a direct interaction with importin-α isoforms. Here we provide a combined nuclear magnetic resonance and small-angle X-ray scattering (NMR/SAXS) analysis to describe the dynamics of the interaction between influenza B NP and the human importin-α. The NP of influenza B does not have a single NLS nor a bipartite NLS but our results suggest that the tail harbors several adjacent NLS sequences, located between residues 30 and 71.

Hierarchical Artificial Bee Colony Algorithm for RFID Network Planning Optimization

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Lianbo Ma

2014-01-01

Full Text Available This paper presents a novel optimization algorithm, namely, hierarchical artificial bee colony optimization, called HABC, to tackle the radio frequency identification network planning (RNP problem. In the proposed multilevel model, the higher-level species can be aggregated by the subpopulations from lower level. In the bottom level, each subpopulation employing the canonical ABC method searches the part-dimensional optimum in parallel, which can be constructed into a complete solution for the upper level. At the same time, the comprehensive learning method with crossover and mutation operators is applied to enhance the global search ability between species. Experiments are conducted on a set of 10 benchmark optimization problems. The results demonstrate that the proposed HABC obtains remarkable performance on most chosen benchmark functions when compared to several successful swarm intelligence and evolutionary algorithms. Then HABC is used for solving the real-world RNP problem on two instances with different scales. Simulation results show that the proposed algorithm is superior for solving RNP, in terms of optimization accuracy and computation robustness.

Hierarchical artificial bee colony algorithm for RFID network planning optimization.

Science.gov (United States)

Ma, Lianbo; Chen, Hanning; Hu, Kunyuan; Zhu, Yunlong

2014-01-01

This paper presents a novel optimization algorithm, namely, hierarchical artificial bee colony optimization, called HABC, to tackle the radio frequency identification network planning (RNP) problem. In the proposed multilevel model, the higher-level species can be aggregated by the subpopulations from lower level. In the bottom level, each subpopulation employing the canonical ABC method searches the part-dimensional optimum in parallel, which can be constructed into a complete solution for the upper level. At the same time, the comprehensive learning method with crossover and mutation operators is applied to enhance the global search ability between species. Experiments are conducted on a set of 10 benchmark optimization problems. The results demonstrate that the proposed HABC obtains remarkable performance on most chosen benchmark functions when compared to several successful swarm intelligence and evolutionary algorithms. Then HABC is used for solving the real-world RNP problem on two instances with different scales. Simulation results show that the proposed algorithm is superior for solving RNP, in terms of optimization accuracy and computation robustness.

Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing in Cryptococcus neoformans and Related Species.

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Wang, Ping

2018-06-27

Cryptococcus neoformans and related species are encapsulated basidiomycetous fungi that cause meningoencephalitis in individuals with immune deficiency. This pathogen has a tractable genetic system; however, gene disruption via electroporation remains difficult, while biolistic transformation is often limited by lack of multiple genetic markers and the high initial cost of equipment. The approach using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has become the technology of choice for gene editing in many organisms due to its simplicity, efficiency, and versatility. The technique has been successfully demonstrated in C. neoformans and Cryptococcus deneoformans in which two DNA plasmids expressing either the Streptococcus pyogenes CAS9 gene or the guide RNA (gRNA) were employed. However, potential adverse effects due to constitutive expression and the time-consuming process of constructing vectors to express each gRNA remain as a primary barrier for wide adaptation. This report describes the delivery of preassembled CRISPR-Cas9-gRNA ribonucleoproteins (RNPs) via electroporation that is able to generate edited mutant alleles. RNP-mediated CRISPR-Cas9 was used to replace the wild-type GIB2 gene encoding a Gβ-like/RACK1 Gib2 protein with a gib2 :: NAT allele via homologous recombination in both C. neoformans and C. deneoformans In addition, a DNA plasmid (pCnCas9:U6-gRNA) that expresses both Cas9 and gRNA, allowing for convenient yet low-cost DNA-mediated gene editing, is described. pCnCas9:U6-gRNA contains an endogenous U6 promoter for gRNA expression and restriction sites for one-step insertion of a gRNA. These approaches and resources provide new opportunities to accelerate genetic studies of Cryptococcus species. IMPORTANCE For genetic studies of the Cryptococcus genus, generation of mutant strains is often hampered by a limited number of selectable genetic markers, the tedious process of vector

Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes.

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Sommerville, John; Brumwell, Craig L; Politz, Joan C Ritland; Pederson, Thoru

2005-03-15

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4

International Nuclear Information System (INIS)

Lorson, Monique A.; Dickson, Alexa M.; Shaw, Debra J.; Todd, Adrian G.; Young, Elizabeth C.; Morse, Robert; Wolstencroft, Catherine; Lorson, Christian L.; Young, Philip J.

2008-01-01

Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex

The management of humeral shaft fractures with associated radial nerve palsy: a review of 117 cases.

Science.gov (United States)

Bumbasirević, Marko; Lesić, Aleksandar; Bumbasirević, Vesna; Cobeljić, Goran; Milosević, Ivan; Atkinson, Henry Dushan E

2010-04-01

This single center retrospective study reviews the management and outcomes of 117 consecutive patients with humeral shaft fractures and associated radial nerve palsy (RNP) treated over a 20-year period (1986-2006). A total of 101 fractures were managed conservatively and 16 fractures underwent external fixation for poor bony alignment. Sixteen grade 1 and 2 open fractures underwent wound toileting alone. No patients underwent initial radial nerve exploration or opening of the fracture sites. All patients achieved clinical and radiological bony union at a mean of 8 weeks (range 7-12 weeks). There were no complications or pin tract infections in the operated patients. A total of 111 cases had initial spontaneous RNP recovery at a mean of 6 weeks (range 3-24 weeks) with full RNP recovery at a mean of 17 weeks (range 3-70 weeks) post-injury. Fourteen patients had no clinical/EMG signs of nerve activity at 12 weeks and 6 subsequently failed to regain any radial nerve recovery; 2 had late explorations and the lacerated nerves underwent sural nerve cable neurorraphy; and 4 patients underwent delayed tendon transposition 2-3 years after initial injury, with good/excellent functional outcomes. Humeral fractures with associated RNP may be treated expectantly. With low rates of humeral nonunion, 95% spontaneous nerve recovery in closed fractures and 94% in grade 1 and 2 open fractures, one has the opportunity of waiting. If at 10-12 weeks there are no clinical/EMG signs of recovery, then nerve exploration/secondary reconstruction is indicated. Late tendon transfers may also give good/excellent functional results.

Individual microRNAs (miRNAs) display distinct mRNA targeting "rules".

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Wang, Wang-Xia; Wilfred, Bernard R; Xie, Kevin; Jennings, Mary H; Hu, Yanling Hu; Stromberg, Arnold J; Nelson, Peter T

2010-01-01

MicroRNAs (miRNAs) guide Argonaute (AGO)-containing microribonucleoprotein (miRNP) complexes to target mRNAs.It has been assumed that miRNAs behave similarly to each other with regard to mRNA target recognition. The usual assumptions, which are based on prior studies, are that miRNAs target preferentially sequences in the 3'UTR of mRNAs,guided by the 5' "seed" portion of the miRNAs. Here we isolated AGO- and miRNA-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with anti-AGO antibody. Cells were transfected with miR-107, miR-124,miR-128, miR-320, or a negative control miRNA. Co-IPed RNAs were subjected to downstream high-density Affymetrix Human Gene 1.0 ST microarray analyses using an assay we validated previously-a "RIP-Chip" experimental design. RIP-Chip data provided a list of mRNAs recruited into the AGO-miRNP in correlation to each miRNA. These experimentally identified miRNA targets were analyzed for complementary six nucleotide "seed" sequences within the transfected miRNAs. We found that miR-124 targets tended to have sequences in the 3'UTR that would be recognized by the 5' seed of miR-124, as described in previous studies. By contrast, miR-107 targets tended to have 'seed' sequences in the mRNA open reading frame, but not the 3' UTR. Further, mRNA targets of miR-128 and miR-320 are less enriched for 6-mer seed sequences in comparison to miR-107 and miR-124. In sum, our data support the importance of the 5' seed in determining binding characteristics for some miRNAs; however, the "binding rules" are complex, and individual miRNAs can have distinct sequence determinants that lead to mRNA targeting.

Multiple Export Mechanisms for mRNAs

Science.gov (United States)

Delaleau, Mildred; Borden, Katherine L. B.

2015-01-01

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

Management of land use land cover through the application of remote sensing, geographic information systems and simulation

Science.gov (United States)

Jha, Praveen

Deforestation and degradation of forest areas, including those in the Protected Areas (PAs), are major concerns in India. There were 2 broad objectives of the study: the technological objective pertained to the development of state-of-art programs that could serve as Decision Support Systems while finalizing plans and policy interventions, while the other objective aimed at generating geo-spatial data in 2 PAs. A part of the Eastern Himalaya biodiversity hotspot, Manas Tiger Reserve (MTR), Assam, India having an area of 2837.12 sq km and an important part of Rajaji-Corbett Tiger Conservation Unit, Rajaji National Park (RNP), Uttarakhand, India, having an area of 820.42 sq km, were taken for the assessment of land use and land cover (LULC) change during 1990--2004. Simulation was undertaken in a smaller area of 1.2 km * 1.2 km right on the fringe of RNP. Three advanced geo-spatial programs---Multi-Algorithm Automation Program (MAAP), Data Automatic Modification Program (DAMP) and Multi-Stage Simulation Program (MUSSIP)---developed by the author were used extensively. Based on the satellite data, MAAP was used for the rapid assessments of LULC of 2004 and 1990; DAMP was used for the spectral modification of the satellite data of the adjacent scenes of 2004 and of 1990; and MUSSIP was used to simulate LULC maps for the future periods (till 2018). These programs produced very high accuracy levels: 91.12% in 2004 and 89.67% in 1990 were obtained for MTR; and 94.87% in 2004 and 94.10% in 1990 were obtained for RNP; 93.40% pixel-to-pixel accuracy and 0.7904 for kappa were achieved for simulation. The annual rate of loss of forests (0.41% in MTR and 1.20% in RNP) and loss of water (1.79% in MTR and 1.69% in RNP) during 1990-2004 is a matter of serious concern. The scenario analysis in the study area for simulation revealed that the deforestation rate of 1.27% per year during 2004--2018 would increase to 2.04% if the human population growth rate is enhanced by 10%. Hence

Zoonotic Risk, Pathogenesis, and Transmission of Avian-Origin H3N2 Canine Influenza Virus.

Science.gov (United States)

Sun, Hailiang; Blackmon, Sherry; Yang, Guohua; Waters, Kaitlyn; Li, Tao; Tangwangvivat, Ratanaporn; Xu, Yifei; Shyu, Daniel; Wen, Feng; Cooley, Jim; Senter, Lucy; Lin, Xiaoxu; Jarman, Richard; Hanson, Larry; Webby, Richard; Wan, Xiu-Feng

2017-11-01

Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes in vitro , and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs. IMPORTANCE IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-09-26

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

The role of Cas8 in type I CRISPR interference.

Science.gov (United States)

Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

2015-05-05

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

CBC bound proteins and RNA fate

DEFF Research Database (Denmark)

Giacometti, Simone

) complex (CBCN), were recently shown to target capped RNA either toward export or degradation, but the mechanisms by which they can discriminate between different RNA families and route them toward different metabolic pathways still remain unclear. A major question to be answered is how and when...... the different CBC subcomplexes are recruited to the RNP. Here, we used an individual nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) approach to identify the transcriptome-wide targets for 5 different components of the CBCAP and CBCN complexes, and compared results to the previously...... analysed NEXT-component RBM7. We report that: (i) CBP20, ARS2, PHAX and ZC3H18 bind close to the cap, while RBM7 and MTR4 bind throughout the mRNA body; (ii) CBP20, ARS2, PHAX and ZC3H18 associate with a broad set of RNA polymerase II (PolII)-derived RNAs and have only mild species preferences; (iii...

Multiobjective RFID Network Optimization Using Multiobjective Evolutionary and Swarm Intelligence Approaches

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Hanning Chen

2014-01-01

Full Text Available The development of radio frequency identification (RFID technology generates the most challenging RFID network planning (RNP problem, which needs to be solved in order to operate the large-scale RFID network in an optimal fashion. RNP involves many objectives and constraints and has been proven to be a NP-hard multi-objective problem. The application of evolutionary algorithm (EA and swarm intelligence (SI for solving multiobjective RNP (MORNP has gained significant attention in the literature, but these algorithms always transform multiple objectives into a single objective by weighted coefficient approach. In this paper, we use multiobjective EA and SI algorithms to find all the Pareto optimal solutions and to achieve the optimal planning solutions by simultaneously optimizing four conflicting objectives in MORNP, instead of transforming multiobjective functions into a single objective function. The experiment presents an exhaustive comparison of three successful multiobjective EA and SI, namely, the recently developed multiobjective artificial bee colony algorithm (MOABC, the nondominated sorting genetic algorithm II (NSGA-II, and the multiobjective particle swarm optimization (MOPSO, on MORNP instances of different nature, namely, the two-objective and three-objective MORNP. Simulation results show that MOABC proves to be more superior for planning RFID networks than NSGA-II and MOPSO in terms of optimization accuracy and computation robustness.

Chinese SLE Treatment and Research Group Registry: III. Association of Autoantibodies with Clinical Manifestations in Chinese Patients with Systemic Lupus Erythematosus

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Jing Li

2014-01-01

Full Text Available We investigated the characteristics of Chinese SLE patients by analyzing the association between specific autoantibodies and clinical manifestations of 2104 SLE patients from registry data of CSTAR cohort. Significant (P<0.05 associations were found between anti-Sm antibody, anti-rRNP antibody, and malar rash; between anti-RNP antibody, anti-SSA antibody, and pulmonary arterial hypertension (PAH; between anti-SSB antibody and hematologic involvement; and between anti-dsDNA antibody and nephropathy. APL antibody was associated with hematologic involvement, interstitial lung disease, and a lower prevalence of oral ulcerations (P<0.05. Associations were also found between anti-dsDNA antibody and a lower prevalence of photosensitivity, and between anti-SSA antibody and a lower prevalence of nephropathy (P<0.05. Most of these findings were consistent with other studies in the literature but this study is the first report on the association between anti-SSA and a lower prevalence of nephropathy. The correlations of specific autoantibodies and clinical manifestations could provide clues for physicians to predict organ damages in SLE patients. We suggest that a thorough screening of autoantibodies should be carried out when the diagnosis of SLE is established, and repeated echocardiography annually in SLE patients with anti-RNP or anti-SSA antibody should be performed.

Long-term bioavailability of redox nanoparticles effectively reduces organ dysfunctions and death in whole-body irradiated mice.

Science.gov (United States)

Feliciano, Chitho P; Tsuboi, Koji; Suzuki, Kenshi; Kimura, Hiroyuki; Nagasaki, Yukio

2017-06-01

Radioprotective agents have been developed to protect patients against the damaging and lethal effects of ionizing radiation. However, in addition to the intrinsic ability to target reactive oxygen species (ROS), the ability to retain a significant level of bioavailability is desirable in radioprotective agents because that would increase and prolong their radioprotective efficacy and improve its safety. Here, we report the development of a novel nanoparticle-based radioprotective agent with improved bioavailability, which suppressed the adverse effects typically associated with low-molecular-weight (LMW) antioxidants. We developed biocompatible and colloidally stable nanoparticles in which nitroxide radicals that were covalently conjugated (redox nanoparticles, RNP N ) effectively scavenged radiation-induced ROS with a characteristically prolonged bioavailability and tissue-residence time compared with that of conventional LMW antioxidants. The confinement of the nitroxide radicals in the RNP N core prevented its rapid metabolism and excretion out of the body. The nano-sized formulation prevented internalization of RNP N in healthy cells, thereby preserving the normal function of the redox reactions in the cell. This improved pharmacological performance dramatically reduced the radiation-induced organ dysfunctions and increased the survival time of the lethally irradiated mice when the nanoparticles were administered 3-24 h before whole-body irradiation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human-like PB2 627K influenza virus polymerase activity is regulated by importin-α1 and -α7.

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Ben Hudjetz

2012-01-01

Full Text Available Influenza A viruses may cross species barriers and transmit to humans with the potential to cause pandemics. Interplay of human- (PB2 627K and avian-like (PB2 627E influenza polymerase complexes with unknown host factors have been postulated to play a key role in interspecies transmission. Here, we have identified human importin-α isoforms (α1 and α7 as positive regulators of human- but not avian-like polymerase activity. Human-like polymerase activity correlated with efficient recruitment of α1 and α7 to viral ribonucleoprotein complexes (vRNPs without affecting subcellular localization. We also observed that human-like influenza virus growth was impaired in α1 and α7 downregulated human lung cells. Mice lacking α7 were less susceptible to human- but not avian-like influenza virus infection. Thus, α1 and α7 are positive regulators of human-like polymerase activity and pathogenicity beyond their role in nuclear transport.

A Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons.

Science.gov (United States)

Vidaki, Marina; Drees, Frauke; Saxena, Tanvi; Lanslots, Erwin; Taliaferro, Matthew J; Tatarakis, Antonios; Burge, Christopher B; Wang, Eric T; Gertler, Frank B

2017-08-02

During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local translation. Copyright © 2017 Elsevier Inc. All rights reserved.

Complex chemistry

International Nuclear Information System (INIS)

Kim, Bong Gon; Kim, Jae Sang; Kim, Jin Eun; Lee, Boo Yeon

2006-06-01

This book introduces complex chemistry with ten chapters, which include development of complex chemistry on history coordination theory and Warner's coordination theory and new development of complex chemistry, nomenclature on complex with conception and define, chemical formula on coordination compound, symbol of stereochemistry, stereo structure and isomerism, electron structure and bond theory on complex, structure of complex like NMR and XAFS, balance and reaction on solution, an organo-metallic chemistry, biology inorganic chemistry, material chemistry of complex, design of complex and calculation chemistry.

Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

Energy Technology Data Exchange (ETDEWEB)

Möhlmann, Sina [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Mathew, Rebecca [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Neumann, Piotr; Schmitt, Andreas [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Lührmann, Reinhard [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Ficner, Ralf, E-mail: rficner@uni-goettingen.de [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany)

2014-06-01

The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

Viral Impact in Autoimmune Diseases: Expanding the “X Chromosome–Nucleolus Nexus” Hypothesis

Science.gov (United States)

Brooks, Wesley H.

2017-01-01

Viruses are suspected of significant roles in autoimmune diseases but the mechanisms are unclear. We get some insight by considering demands a virus places on host cells. Viruses not only require production of their own proteins, RNA and/or DNA, but also production of additional cellular machinery, such as ribosomes, to handle the increased demands. Since the nucleolus is a major site of RNA processing and ribonucleoprotein assembly, nucleoli are targeted by viruses, directly when viral RNA and proteins enter the nucleolus and indirectly when viruses induce increased expression of cellular polyamine genes. Polyamines are at high levels in nucleoli to assist in RNA folding. The size and activity of nucleoli increase directly with increases in polyamines. Nucleolar expansion due to abnormal increases in polyamines could disrupt nearby chromatin, such as the inactive X chromosome, leading to expression of previously sequestered DNA. Sudden expression of a large concentration of Alu elements from the disrupted inactive X can compete with RNA transcripts containing intronic Alu sequences that normally maintain nucleolar structural integrity. Such disruption of nucleolar activity can lead to misfolded RNAs, misassembled ribonucleoprotein complexes, and fragmentation of the nucleolus. Many autoantigens in lupus are, at least transiently, components of the nucleolus. Considering these effects of viruses, the “X chromosome–nucleolus nexus” hypothesis, which proposed disruption of the inactive X by the nucleolus during stress, is now expanded here to propose subsequent disruption of the nucleolus by previously sequestered Alu elements, which can fragment the nucleolus, leading to generation of autoantigens. PMID:29234321

Autoimmunity due to molecular mimicry as a cause of neurological disease.

Science.gov (United States)

Levin, Michael C; Lee, Sang Min; Kalume, Franck; Morcos, Yvette; Dohan, F Curtis; Hasty, Karen A; Callaway, Joseph C; Zunt, Joseph; Desiderio, Dominic; Stuart, John M

2002-05-01

One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

Student perception of the educational environment in regular and bridging nursing programs in Saudi Arabia using the Dundee Ready Educational Environment Measure.

Science.gov (United States)

Al Nozha, Omar Mansour; Fadel, Hani T

2017-01-01

Taibah University offers regular nursing (RNP) and nursing bridging (NBP) bachelor programs. We evaluated student perception of the learning environment as one means of quality assurance. To assess nursing student perception of their educational environment, to compare the perceptions of regular and bridging students, and to compare the perceptions of students in the old and new curricula. Cross-sectional survey. College of Nursing at Taibah University, Madinah, Saudi Arabia. The Dundee Ready Educational Environment Measure (DREEM) instrument was distributed to over 714 nursing students to assess perception of the educational environment. Independent samples t test and Pearson's chi square were used to compare the programs and curricula. The DREEM inventory score. Of 714 students, 271 (38%) were RNP students and 443 (62%) were NBP students. The mean (standard deviation) DREEM score was 111 (25). No significant differences were observed between the programs except for the domain "academic self-perceptions" being higher in RNP students (P .05). Nursing students generally perceived their learning environment as more positive than negative. Regular students were more positive than bridging students. Students who experienced the new curriculum were more positive towards learning. The cross-sectional design and unequal gender and study level distributions may limit generalizability of the results. Longitudinal, large-scale studies with more even distributions of participant characteristics are needed.

Enzyme-linked immunosorbent assay (ELISA for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests Reação imunoenzimática (ELISA para detecção de anticorpos para o vírus do sarampo: comparação com reações de inibição da hema-glutinação, imunofluorescência indireta e neutralização de placas

Directory of Open Access Journals (Sweden)

Vanda Akico Ueda Fick de Souza

1991-02-01

Full Text Available An enzyme-linked immunosorbent assay (ELISA for measles antibodies was compared with Plaque Neutralization (PRN, Haemagglutination inhibition (HI and Fluorescent antibody (IFA tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%. IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.A reação imunoenzimática (ELISA para determinação de anticorpos para o vírus do sarampo foi comparada com a reação de neutralização de placas (RNP, inibição da hemaglutinação (RIH e imunofluorescência indireta (RIF. Das 179 amostras positivas pela RNP, somente 2, com títulos iguais a 8, se apresentaram negativas por ELISA (copositividade de 98.9%. A RIF e RIH apresentaram, respectivamente, copositividade de 93.3 e 82.7%. ELISA apresentou sensibilidade equivalente à complexa RNP, boa reprodutibilidade e representa uma alternativa para a detecção de baixos títulos de anticorpos contra o sarampo.

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Indian Academy of Sciences (India)

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Two component system. The Kdp-ATPase ... Heterogeneous nuclear ribonucleoprotein D/AUF1 interacts ... Alexe G. Analysis of breast cancer progression using principal compo- ... netic trees of homologous proteins: Inferences on protein.

75 FR 60304 - Standard Instrument Approach Procedures, and Takeoff Minimums and Obstacle Departure Procedures...

Science.gov (United States)

2010-09-30

... are available online free of charge. Visit http://www.nfdc.faa.gov to register. Additionally... (RNP) Z RWY 26, Amdt 1 Rifle, CO, Garfield County Rgnl, SQUAT THREE GRAPHIC OBSTACLE DP Rifle, CO...

Human factors research plan for instrument procedures : FY12 version 1.1

Science.gov (United States)

2012-06-19

This research will support the development of instrument procedures for performance-based navigation (PBN) operations. These procedures include, but are not limited to, area navigation (RNAV) and required navigation performance (RNP) operations. The ...

Mixed connective tissue disease: The King Faisal Specialist Hospital experience

International Nuclear Information System (INIS)

Al-Rayes, H.; Al-Sheikh, A.; Al-Dalaan, A.; Al-Saleh, S.

2002-01-01

The aim of this study was to assess the clinical presentation, complications and serological analysis of mixed connective tissue disease (MCTD) at King Faisal Specialist Hospital and Research Centre (KFSHRC), and to determine the long-term clinical and immunologic outcomes. This was a retrospective study with prospective follow-up of 18 patients with MCTD who were followed at KFSHRC between 1982 and 1999. The age at onset of the disease ranged from 6 to 44 years, with mean age of 17.9 years. The female to male ratio was 2.5:1 and the mean follow-up time was 5 years. The most frequent presenting symptoms were arthralgia in all patients, Raynaud's phenomenon in 16 patients (88%) and swollen hands in 11 patients (61%). Arthritis was seen in 12 patients in (67%) and definite myositis in 10 patients (58%). The most common skin rashes encountered included lupus-like rash in 8 patients (44%) and cutaneous vasculitis in 5 patients (28%). Pulmonary hypertension occurred in 4 patients (22%). Other clinical manifestations encountered were esophageal hypomotility in 10 patients (56%), myocarditis in 2 patients (11%) and proteinurea in 2 patients (11%), while various neurological manifestations were present in 7 patients (39%). All patients exhibited higher titer of ANA and anti-nRNP antibodies. Five of the 18 patients (28%) had marked reduction in the anti-nRNP during remission. Following treatment, features of inflammation as well as Raynaud's phenomenon and esophageal hypomotility diminished, while pulmonary hypertension persisted. A favorable outcome was observed in 12 patients (67%), 3 patients (17%) had continued active disease, while 3 patients (17%) died, with death related to pulmonary hypertension occurring in 2 patients (11%). The studied patients demonstrated the typical clinical and serological findings of MCTD, which support the correlation between anti-nRNP antibody specificities and MCTD. Autoantibody reactivity against nRNP polypeptides tends to regress during

ORF Sequence: NC_001134 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ivery of heterogeneous nuclear ribonucleoproteins to the nucleoplasm, binds rg-nucl... NC_001134 gi|6319491 >gi|6319491|ref|NP_009573.1| Transportin, cytosolic karyopherin beta 2 involved in del

ComplexViewer: visualization of curated macromolecular complexes.

Science.gov (United States)

Combe, Colin W; Sivade, Marine Dumousseau; Hermjakob, Henning; Heimbach, Joshua; Meldal, Birgit H M; Micklem, Gos; Orchard, Sandra; Rappsilber, Juri

2017-11-15

Proteins frequently function as parts of complexes, assemblages of multiple proteins and other biomolecules, yet network visualizations usually only show proteins as parts of binary interactions. ComplexViewer visualizes interactions with more than two participants and thereby avoids the need to first expand these into multiple binary interactions. Furthermore, if binding regions between molecules are known then these can be displayed in the context of the larger complex. freely available under Apache version 2 license; EMBL-EBI Complex Portal: http://www.ebi.ac.uk/complexportal; Source code: https://github.com/MICommunity/ComplexViewer; Package: https://www.npmjs.com/package/complexviewer; http://biojs.io/d/complexviewer. Language: JavaScript; Web technology: Scalable Vector Graphics; Libraries: D3.js. colin.combe@ed.ac.uk or juri.rappsilber@ed.ac.uk. © The Author 2017. Published by Oxford University Press.

Comparison of indirect immunofluorescence test for measles antibodies with haemagglutination inhibition and plaque neutralization tests Comparação da reação de imunofluorescência indireta para o vírus do sarampo com as reações de inibição da hemaglutinação e neutralização por redução de placas

Directory of Open Access Journals (Sweden)

Vanda Akico Ueda Fick de Souza

1990-10-01

Full Text Available Indirect Immunofluorescence (IFA, Plaque Reduction Neutralization (PRN and Haemagglutination Inhibition (HI tests for measles antibodies were carried out in 197 sera obtained from umbilical cord and vaccinated children. The IFA was also applied to blood samples collected with filter paper. IFA results demonstrated that the test is relatively simple to perform, with good reproducibility for different antigen lots. Good correlation was obtained between IFA, PRN and HI antibody titers. Better correlation was demonstrated with IFA and PRN than with HI and PRN tests. Sensitivity of IFA in detecting antibody was less effective than PRN, however more effective than HI using rhesus monkey red blood cells. PRN antibody titers over 100 were detected by IFA but not by HI (9.7% with negative results. IFA may be of considerable practical use and able to substitute HI in Seroepidemiological surveys and to evaluate vaccine efficacy. It also can be simplified by employing filter paper collected samples.As reações de imunofluorescência indireta (RIF, neutralização por redução de placas (RNP e inibição da hemaglutinação (RIH para detecção de anticorpos para o vírus do sarampo foram aplicadas a 197 soros provenientes de cordão umbilical e de crianças vacinadas contra o sarampo. Avaliou-se ainda a aplicação da RIF em amostras colhidas em papel de filtro. A RIF apresentou-se como uma prova de execução relativamente simples, de boa reprodutibilidade com diferentes partidas de antígeno. Observou-se boa correlação entre os títulos de anticorpos obtidos por RIF, RNP e RIH. Com a RNP, a RIF apresentou maior correlação que a RIH. A sensibilidade da RIF na detecção de anticorpos contra o sarampo foi menor que RNP, porém superior à da RIH com hemácias de macaco rhesus. Anticorpos com títulos superiores a 100 pela RNP foram sistematicamente detectados pela RIF, mas não por RIH (9,7% de resultados negativos. A RIF pode ser de grande utilidade

Synchronization in node of complex networks consist of complex chaotic system

Energy Technology Data Exchange (ETDEWEB)

Wei, Qiang, E-mail: qiangweibeihua@163.com [Beihua University computer and technology College, BeiHua University, Jilin, 132021, Jilin (China); Digital Images Processing Institute of Beihua University, BeiHua University, Jilin, 132011, Jilin (China); Faculty of Electronic Information and Electrical Engineering, Dalian University of Technology, Dalian, 116024 (China); Xie, Cheng-jun [Beihua University computer and technology College, BeiHua University, Jilin, 132021, Jilin (China); Digital Images Processing Institute of Beihua University, BeiHua University, Jilin, 132011, Jilin (China); Liu, Hong-jun [School of Information Engineering, Weifang Vocational College, Weifang, 261041 (China); Li, Yan-hui [The Library, Weifang Vocational College, Weifang, 261041 (China)

2014-07-15

A new synchronization method is investigated for node of complex networks consists of complex chaotic system. When complex networks realize synchronization, different component of complex state variable synchronize up to different scaling complex function by a designed complex feedback controller. This paper change synchronization scaling function from real field to complex field for synchronization in node of complex networks with complex chaotic system. Synchronization in constant delay and time-varying coupling delay complex networks are investigated, respectively. Numerical simulations are provided to show the effectiveness of the proposed method.

Characterization of MRP RNA-protein interactions within the perinucleolar compartment.

Science.gov (United States)

Pollock, Callie; Daily, Kelly; Nguyen, Van Trung; Wang, Chen; Lewandowska, Marzena Anna; Bensaude, Olivier; Huang, Sui

2011-03-15

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA-processing (MRP) RNA, pyrimidine tract-binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA-containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)-PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA-protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC.

Genome-wide identification and functional prediction of novel and fungi-responsive lincRNAs in Triticum aestivum.

Science.gov (United States)

Zhang, Hong; Hu, Weiguo; Hao, Jilei; Lv, Shikai; Wang, Changyou; Tong, Wei; Wang, Yajuan; Wang, Yanzhen; Liu, Xinlun; Ji, Wanquan

2016-03-15

Stripe rust (Puccinia striiformis f. sp. tritici; Pst) and powdery mildew (Blumeria graminis f. sp. tritici; Bgt) are important diseases of wheat (Triticum aestivum) worldwide. Increasingly evidences suggest that long intergenic ncRNAs (lincRNAs) are developmentally regulated and play important roles in development and stress responses of plants. However, identification of lincRNAs in wheat is still limited comparing with functional gene expression. The transcriptome of the hexaploid wheat line N9134 inoculated with the Chinese Pst race CYR31 and Bgt race E09 at 1, 2, and 3 days post-inoculation was recapitulated to detect the lincRNAs. Here, 283 differential expressed lincRNAs were identified from 58218 putative lincRNAs, which account for 31.2% of transcriptome. Of which, 254 DE-LincRNAs responded to the Bgt stress, and 52 lincRNAs in Pst. Among them, 1328 SnRNP motifs (sm sites) were detected and showed RRU4-11RR sm site element and consensus RRU1-9VU1-7RR SnRNP motifs, where the total number of uridine was more than 3 but less than 11. Additionally, 101 DE-lincRNAs were predicted as targets of miRNA by psRNATarget, while 5 target mimics were identified using target mimicry search in TAPIR. Taken together, our findings indicate that the lincRNA of wheat responded to Bgt and Pst stress and played important roles in splicesome and inter-regulating with miRNA. The sm site of wheat showed a more complex construction than that in mammal and model plant. The mass sequence data generated in this study provide a cue for future functional and molecular research on wheat-fungus interactions.

Generalized Combination Complex Synchronization for Fractional-Order Chaotic Complex Systems

Directory of Open Access Journals (Sweden)

Cuimei Jiang

2015-07-01

Full Text Available Based on two fractional-order chaotic complex drive systems and one fractional-order chaotic complex response system with different dimensions, we propose generalized combination complex synchronization. In this new synchronization scheme, there are two complex scaling matrices that are non-square matrices. On the basis of the stability theory of fractional-order linear systems, we design a general controller via active control. Additionally, by virtue of two complex scaling matrices, generalized combination complex synchronization between fractional-order chaotic complex systems and real systems is investigated. Finally, three typical examples are given to demonstrate the effectiveness and feasibility of the schemes.

snRNP proteins in health and disease

Czech Academy of Sciences Publication Activity Database

Krausová, Michaela; Staněk, David

S1084-9521, č. 17 (2017), s. 30150-30157 ISSN 1084-9521 R&D Projects: GA ČR GA15-00790S; GA MŠk LO1419 Institutional support: RVO:68378050 Keywords : Congenital craniofacial disorders * Haematological malignancies * Mutations * Retinopathy * Spliceosome Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 6.614, year: 2016

Transcription-associated quality control of mRNP

DEFF Research Database (Denmark)

Schmid, Manfred; Jensen, Torben Heick

2013-01-01

Although a prime purpose of transcription is to produce RNA, a substantial amount of transcript is nevertheless turned over very early in its lifetime. During transcription RNAs are matured by nucleases from longer precursors and activities are also employed to exert quality control over the RNA...

Complexity explained

CERN Document Server

Erdi, Peter

2008-01-01

This book explains why complex systems research is important in understanding the structure, function and dynamics of complex natural and social phenomena. Readers will learn the basic concepts and methods of complex system research.

On Measuring the Complexity of Networks: Kolmogorov Complexity versus Entropy

Directory of Open Access Journals (Sweden)

Mikołaj Morzy

2017-01-01

Full Text Available One of the most popular methods of estimating the complexity of networks is to measure the entropy of network invariants, such as adjacency matrices or degree sequences. Unfortunately, entropy and all entropy-based information-theoretic measures have several vulnerabilities. These measures neither are independent of a particular representation of the network nor can capture the properties of the generative process, which produces the network. Instead, we advocate the use of the algorithmic entropy as the basis for complexity definition for networks. Algorithmic entropy (also known as Kolmogorov complexity or K-complexity for short evaluates the complexity of the description required for a lossless recreation of the network. This measure is not affected by a particular choice of network features and it does not depend on the method of network representation. We perform experiments on Shannon entropy and K-complexity for gradually evolving networks. The results of these experiments point to K-complexity as the more robust and reliable measure of network complexity. The original contribution of the paper includes the introduction of several new entropy-deceiving networks and the empirical comparison of entropy and K-complexity as fundamental quantities for constructing complexity measures for networks.

Complex dynamical invariants for two-dimensional complex potentials

Indian Academy of Sciences (India)

Abstract. Complex dynamical invariants are searched out for two-dimensional complex poten- tials using rationalization method within the framework of an extended complex phase space characterized by x = x1 + ip3, y = x2 + ip4, px = p1 + ix3, py = p2 + ix4. It is found that the cubic oscillator and shifted harmonic oscillator ...

Complex Fuzzy Set-Valued Complex Fuzzy Measures and Their Properties

Science.gov (United States)

Ma, Shengquan; Li, Shenggang

2014-01-01

Let F*(K) be the set of all fuzzy complex numbers. In this paper some classical and measure-theoretical notions are extended to the case of complex fuzzy sets. They are fuzzy complex number-valued distance on F*(K), fuzzy complex number-valued measure on F*(K), and some related notions, such as null-additivity, pseudo-null-additivity, null-subtraction, pseudo-null-subtraction, autocontionuous from above, autocontionuous from below, and autocontinuity of the defined fuzzy complex number-valued measures. Properties of fuzzy complex number-valued measures are studied in detail. PMID:25093202

The complex portal--an encyclopaedia of macromolecular complexes.

Science.gov (United States)

Meldal, Birgit H M; Forner-Martinez, Oscar; Costanzo, Maria C; Dana, Jose; Demeter, Janos; Dumousseau, Marine; Dwight, Selina S; Gaulton, Anna; Licata, Luana; Melidoni, Anna N; Ricard-Blum, Sylvie; Roechert, Bernd; Skyzypek, Marek S; Tiwari, Manu; Velankar, Sameer; Wong, Edith D; Hermjakob, Henning; Orchard, Sandra

2015-01-01

The IntAct molecular interaction database has created a new, free, open-source, manually curated resource, the Complex Portal (www.ebi.ac.uk/intact/complex), through which protein complexes from major model organisms are being collated and made available for search, viewing and download. It has been built in close collaboration with other bioinformatics services and populated with data from ChEMBL, MatrixDB, PDBe, Reactome and UniProtKB. Each entry contains information about the participating molecules (including small molecules and nucleic acids), their stoichiometry, topology and structural assembly. Complexes are annotated with details about their function, properties and complex-specific Gene Ontology (GO) terms. Consistent nomenclature is used throughout the resource with systematic names, recommended names and a list of synonyms all provided. The use of the Evidence Code Ontology allows us to indicate for which entries direct experimental evidence is available or if the complex has been inferred based on homology or orthology. The data are searchable using standard identifiers, such as UniProt, ChEBI and GO IDs, protein, gene and complex names or synonyms. This reference resource will be maintained and grow to encompass an increasing number of organisms. Input from groups and individuals with specific areas of expertise is welcome. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Complexity Plots

KAUST Repository

Thiyagalingam, Jeyarajan

2013-06-01

In this paper, we present a novel visualization technique for assisting the observation and analysis of algorithmic complexity. In comparison with conventional line graphs, this new technique is not sensitive to the units of measurement, allowing multivariate data series of different physical qualities (e.g., time, space and energy) to be juxtaposed together conveniently and consistently. It supports multivariate visualization as well as uncertainty visualization. It enables users to focus on algorithm categorization by complexity classes, while reducing visual impact caused by constants and algorithmic components that are insignificant to complexity analysis. It provides an effective means for observing the algorithmic complexity of programs with a mixture of algorithms and black-box software through visualization. Through two case studies, we demonstrate the effectiveness of complexity plots in complexity analysis in research, education and application. © 2013 The Author(s) Computer Graphics Forum © 2013 The Eurographics Association and Blackwell Publishing Ltd.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

Science.gov (United States)

Brosius, Jürgen

2014-01-01

Currently, the best scenario for earliest forms of life is based on RNA molecules as they have the proven ability to catalyze enzymatic reactions and harbor genetic information. Evolutionary principles valid today become apparent in such models already. Furthermore, many features of eukaryotic genome architecture might have their origins in an RNA or RNA/protein (RNP) world, including the onset of a further transition, when DNA replaced RNA as the genetic bookkeeper of the cell. Chromosome maintenance, splicing, and regulatory function via RNA may be deeply rooted in the RNA/RNP worlds. Mostly in eukaryotes, conversion from RNA to DNA is still ongoing, which greatly impacts the plasticity of extant genomes. Raw material for novel genes encoding protein or RNA, or parts of genes including regulatory elements that selection can act on, continues to enter the evolutionary lottery. PMID:25081515

Induction of a systemic lupus erythematosus-like disease in mice by a common human anti-DNA idiotype

International Nuclear Information System (INIS)

Mendlovic, S.; Brocke, S.; Meshorer, A.; Mozes, E.; Shoenfeld, Y.; Bakimer, R.; Ben-Bassat, M.

1988-01-01

Systemic lupus erythematosus (SLE) is considered to be the quintessential autoimmune disease. It has not been possible to induce SLE in animal models by DNA immunization or by challenge with anti-DNA antibodies. The authors report a murine model of SLE-like disease induced by immunization of C3H.SW female mice with a common human monoclonal anti-DNA idiotype (16/6 idiotype). Following a booster injection with the 16/6 idiotype, high levels of murine anti-16/6 and anti-anti-16/6 antibodies (associated with anti-DNA activity) were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with DNA, poly(I), poly(dT), ribonucleoprotein, autoantigens [Sm, SS-A (Ro), and SS-B (La)], and cardiolipin were noted. The serological findings were associated with increased erythrocyte sedimentation rate, leukopenia, proteinuria, immune complex deposition in the glomerular mesangium, and sclerosis of the glomeruli. The immune complexes in the kidneys were shown to contain the 16/6 idiotype. This experimental SLE-like model may be used to elucidate the mechanisms underlying SLE

RNA search engines empower the bacterial intranet.

Science.gov (United States)

Dendooven, Tom; Luisi, Ben F

2017-08-15

RNA acts not only as an information bearer in the biogenesis of proteins from genes, but also as a regulator that participates in the control of gene expression. In bacteria, small RNA molecules (sRNAs) play controlling roles in numerous processes and help to orchestrate complex regulatory networks. Such processes include cell growth and development, response to stress and metabolic change, transcription termination, cell-to-cell communication, and the launching of programmes for host invasion. All these processes require recognition of target messenger RNAs by the sRNAs. This review summarizes recent results that have provided insights into how bacterial sRNAs are recruited into effector ribonucleoprotein complexes that can seek out and act upon target transcripts. The results hint at how sRNAs and their protein partners act as pattern-matching search engines that efficaciously regulate gene expression, by performing with specificity and speed while avoiding off-target effects. The requirements for efficient searches of RNA patterns appear to be common to all domains of life. © 2017 The Author(s).

Complex differential geometry

CERN Document Server

Zheng, Fangyang

2002-01-01

The theory of complex manifolds overlaps with several branches of mathematics, including differential geometry, algebraic geometry, several complex variables, global analysis, topology, algebraic number theory, and mathematical physics. Complex manifolds provide a rich class of geometric objects, for example the (common) zero locus of any generic set of complex polynomials is always a complex manifold. Yet complex manifolds behave differently than generic smooth manifolds; they are more coherent and fragile. The rich yet restrictive character of complex manifolds makes them a special and interesting object of study. This book is a self-contained graduate textbook that discusses the differential geometric aspects of complex manifolds. The first part contains standard materials from general topology, differentiable manifolds, and basic Riemannian geometry. The second part discusses complex manifolds and analytic varieties, sheaves and holomorphic vector bundles, and gives a brief account of the surface classifi...

(II) complexes

African Journals Online (AJOL)

activities of Schiff base tin (II) complexes. Neelofar1 ... Conclusion: All synthesized Schiff bases and their Tin (II) complexes showed high antimicrobial and ...... Singh HL. Synthesis and characterization of tin (II) complexes of fluorinated Schiff bases derived from amino acids. Spectrochim Acta Part A: Molec Biomolec.

Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs).

Science.gov (United States)

Vazquez-Martin, Alejandro; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Menendez, Javier A

2011-09-15

Raptor is the key scaffolding protein that recruits mTOR substrates to rapamycin-sensitive mTOR complex 1 (mTORC1), a molecular integrator of mitogenic and nutrient/energy environmental inputs into protein translation and cell growth. Although Raptor phosphorylation on various sites is pivotal in the regulation of mTORC1 activity, it remains to be elucidated whether site-specific phosphorylation differentially distributes Raptor to unique subcellular compartments. When exploring the spatiotemporal cell cycle dynamics of six different phospho (P)-Raptor isoforms (Thr ( 706) , Ser ( 722) , Ser ( 863) , Ser ( 792) and Ser ( 877) ), a number of remarkable events differentially defined a topological resetting of P-RaptorThr706 on interphasic and mitotic chromosomes. In interphase nuclei, P-Raptor (Thr706) co-localized with fibrillarin, a component of the nucleolar small nuclear ribonucleoprotein particle, as well as with RNA polymerase I, the enzyme that transcribes nucleolar rRNA. Upon Actinomycin D-induced nucleolar segregation and disaggregation, P-RaptorThr706 was excluded from the nucleolus to accumulate at discrete nucleoplasmic bodies. During mitosis, CDK1 inhibition-induced premature assembly of nucleoli relocated fibrillarin to the surrounding regions of chromosomal-associated P-Raptor (Thr706) , suggesting that a subpopulation of mitotic P-Raptor (Thr706) remained targeted at chromosomal loops of rDNA or nuclear organizer regions (NORs). At the end of mitosis and cytokinesis, when reassembly of incipient nucleoli begins upon NORs activation of rDNA transcription, fibrillarin spatially reorganized with P-Raptor (Thr706) to give rise to daughter nucleoli. Treatment with IGF1 exclusively hyperactivated nuclear P-Raptor (Ser706) and concomitantly promoted Ser ( 2481) autophosphorylation of mTOR, which monitors mTORC1-associated catalytic activity. Nucleolar- and NOR-associated P-Raptor (Ser706) may physically link mTORC1 signaling to ever-growing nucleolus

Workshop on Recommendation in Complex Scenarios (ComplexRec 2017)

DEFF Research Database (Denmark)

Bogers, Toine; Koolen, Marijn; Mobasher, Bamshad

2017-01-01

Recommendation algorithms for ratings prediction and item ranking have steadily matured during the past decade. However, these state-of-the-art algorithms are typically applied in relatively straightforward scenarios. In reality, recommendation is often a more complex problem: it is usually just...... a single step in the user's more complex background need. These background needs can often place a variety of constraints on which recommendations are interesting to the user and when they are appropriate. However, relatively little research has been done on these complex recommendation scenarios....... The ComplexRec 2017 workshop addressed this by providing an interactive venue for discussing approaches to recommendation in complex scenarios that have no simple one-size-fits-all-solution....

A importância da contra-imunoeletroforese na detecção de antígenos nucleares extraíveis para o diagnóstico de doenças reumáticas sistêmicas The importance of counterimmunoelectrophoresis in the detection of extractable nuclear antigens for the diagnosis of systemic rheumatic diseases

Directory of Open Access Journals (Sweden)

Rita de Cassia Siqueira Bruder

2004-02-01

Full Text Available Os antígenos nucleares extraíveis (ENAs são encontrados no soro da maioria dos pacientes com doença reumática sistêmica. Os principais ENAs estudados são SS-A/Ro, SS-B/La, RNP, Sm, Scl-70 e Jo-1. Objeti-vou-se neste trabalho: a padronizar a técnica de contra-imunoeletroforese (CIE para a detecção de ENAs; b padronizar o substrato antigênico (ENAs para a CIE a partir de baço de cão; c comparar os resultados da CIE com as técnicas de imunofluorescência indireta (IFI e Elisa para esses antígenos. Para tal foram estudados 40 soros de pacientes com doença reumática sistêmica confirmada por exames clínico e laboratorial (sorológico e biópsia. Como controle negativo foram utilizados dez soros de doadores de sangue normais, e, como controles positivos, seis soros-padrão anti-Ro, anti-La, anti-RNP, anti-Sm, anti-Scl-70, anti-Jo-1, para caracterizar os antígenos presentes no extrato de baço. Neste último foram detectados vários ENAs, exceto RNP e Scl-70. A técnica de CIE apresentou boas sensibilidade (70% e especificidade (100% em relação às outras técnicas (IFI e Elisa. A titulação dos soros pela CIE revelou positividade até diluições de 1:16 em 32,5% dos casos. Concluímos que a CIE e os antígenos extraídos de baço de cão podem ser utilizados na rotina laboratorial para triagem destes ENAs, com a vantagem, em relação à IFI, de poderem ser titulados.Extractable nuclear antigens (ENAs are detected in the sera of the majority of patients with rheumatic systemic disease. The main ENAs studied are SS-A/Ro, SS-B/La, RNP, Sm, Scl-70 and Jo-1. The aim of this work was: a to standardize the counterimmunoelectrophoresis technique (CIE to detect ENAs; b to standardize the extration of ENAs from dog spleen as a substract for CIE test; c to compare the results obtained by CIE with those of indirect immunofluorescence (IFI and Elisa for these antigens. Forty sera from individuals with rheumatic systemic disease confirmed by

THE RELATIONSHIP BETWEEN AUTOIMMUNE ANTIBODIES AND HCG TREATMENT 1N HABITUAL ABORTION

Institute of Scientific and Technical Information of China (English)

TANGPei-Zhong; WUJin-Zhi; BAOChun-De; CHENShun-Le

1989-01-01

The antibodies to cardiolipin (aCL), double stranded DNA (aDNA) and to nuclear axttigcns(Sm, SSA, SSB, Ribonucleoprotein) were prospe, ctivcly investigated in 86 patients of habitual abortion without abilormaiity in their reprodutive system and karyotypes. All

Dynamic complexity: plant receptor complexes at the plasma membrane.

Science.gov (United States)

Burkart, Rebecca C; Stahl, Yvonne

2017-12-01

Plant receptor complexes at the cell surface perceive many different external and internal signalling molecules and relay these signals into the cell to regulate development, growth and immunity. Recent progress in the analyses of receptor complexes using different live cell imaging approaches have shown that receptor complex formation and composition are dynamic and take place at specific microdomains at the plasma membrane. In this review we focus on three prominent examples of Arabidopsis thaliana receptor complexes and how their dynamic spatio-temporal distribution at the PM has been studied recently. We will elaborate on the newly emerging concept of plasma membrane microdomains as potential hubs for specific receptor complex assembly and signalling outputs. Copyright © 2017 Elsevier Ltd. All rights reserved.

The mechanism of vault opening from the high resolution structure of the N-terminal repeats of MVP.

Science.gov (United States)

Querol-Audí, Jordi; Casañas, Arnau; Usón, Isabel; Luque, Daniel; Castón, José R; Fita, Ignasi; Verdaguer, Nuria

2009-11-04

Vaults are ubiquitous ribonucleoprotein complexes involved in a diversity of cellular processes, including multidrug resistance, transport mechanisms and signal transmission. The vault particle shows a barrel-shaped structure organized in two identical moieties, each consisting of 39 copies of the major vault protein MVP. Earlier data indicated that vault halves can dissociate at acidic pH. The crystal structure of the vault particle solved at 8 A resolution, together with the 2.1-A structure of the seven N-terminal domains (R1-R7) of MVP, reveal the interactions governing vault association and provide an explanation for a reversible dissociation induced by low pH. The structural comparison with the recently published 3.5 A model shows major discrepancies, both in the main chain tracing and in the side chain assignment of the two terminal domains R1 and R2.

Revitalizing Complex Analysis: A Transition-to-Proof Course Centered on Complex Topics

Science.gov (United States)

Sachs, Robert

2017-01-01

A new transition course centered on complex topics would help in revitalizing complex analysis in two ways: first, provide early exposure to complex functions, sparking greater interest in the complex analysis course; second, create extra time in the complex analysis course by eliminating the "complex precalculus" part of the course. In…

3D complex: a structural classification of protein complexes.

Directory of Open Access Journals (Sweden)

Emmanuel D Levy

2006-11-01

Full Text Available Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

Unraveling chaotic attractors by complex networks and measurements of stock market complexity

International Nuclear Information System (INIS)

Cao, Hongduo; Li, Ying

2014-01-01

We present a novel method for measuring the complexity of a time series by unraveling a chaotic attractor modeled on complex networks. The complexity index R, which can potentially be exploited for prediction, has a similar meaning to the Kolmogorov complexity (calculated from the Lempel–Ziv complexity), and is an appropriate measure of a series' complexity. The proposed method is used to research the complexity of the world's major capital markets. None of these markets are completely random, and they have different degrees of complexity, both over the entire length of their time series and at a level of detail. However, developing markets differ significantly from mature markets. Specifically, the complexity of mature stock markets is stronger and more stable over time, whereas developing markets exhibit relatively low and unstable complexity over certain time periods, implying a stronger long-term price memory process

Unraveling chaotic attractors by complex networks and measurements of stock market complexity.

Science.gov (United States)

Cao, Hongduo; Li, Ying

2014-03-01

We present a novel method for measuring the complexity of a time series by unraveling a chaotic attractor modeled on complex networks. The complexity index R, which can potentially be exploited for prediction, has a similar meaning to the Kolmogorov complexity (calculated from the Lempel-Ziv complexity), and is an appropriate measure of a series' complexity. The proposed method is used to research the complexity of the world's major capital markets. None of these markets are completely random, and they have different degrees of complexity, both over the entire length of their time series and at a level of detail. However, developing markets differ significantly from mature markets. Specifically, the complexity of mature stock markets is stronger and more stable over time, whereas developing markets exhibit relatively low and unstable complexity over certain time periods, implying a stronger long-term price memory process.

Translations on USSR Science and Technology, Biomedical and Behavioral Sciences, Number 42

Science.gov (United States)

1978-08-08

56 Strain; b—Radioactivity Distribution, Following Centrifugation (in the same conditions), of a Total RNP Preparation Isolated from the Bio ...their metabolites, rogor and DDVP in the environment, when these agents are used in orchard growing and viticulture . The orchard was sprayed with

75 FR 9095 - Standard Instrument Approach Procedures, and Takeoff Minimums and Obstacle Departure Procedures...

Science.gov (United States)

2010-03-01

... Hayden, CO, Yampa Valley, ILS OR LOC/DME Y RWY 10, Amdt 3 Hayden, CO, Yampa Valley, RNAV (GPS) RWY 28, Amdt 1 Hayden, CO, Yampa Valley, RNAV (GPS) Y RWY 10, Amdt 2 Hayden, CO, Yampa Valley, RNAV (RNP) Z RWY...

Experiment list: SRX107407 [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available osis=Adenocarcinoma 37670219,66.5,15.7,675 GSM838385: hnrnpl sictrl ChIP-Seq; Homo sapiens; ChIP-Seq source_name=Hela cells knock...down control || chip antibody=hnRNP L || treatment=knockdown control || cell line=HeLa

Promoter proximal polyadenylation sites reduce transcription activity

DEFF Research Database (Denmark)

Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

2012-01-01

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site......, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...... on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

Science.gov (United States)

Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

2014-11-01

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people

High-resolution NMR structures of the domains of Saccharomyces cerevisiae Tho1

International Nuclear Information System (INIS)

Jacobsen, Julian O. B.; Allen, Mark D.; Freund, Stefan M. V.; Bycroft, Mark

2016-01-01

In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and the C-terminal RNA-binding domain of S. cerevisiae Tho1 have been determined. THO is a multi-protein complex involved in the formation of messenger ribonuclear particles (mRNPs) by coupling transcription with mRNA processing and export. THO is thought to be formed from five subunits, Tho2p, Hpr1p, Tex1p, Mft1p and Thp2p, and recent work has determined a low-resolution structure of the complex [Poulsen et al. (2014 ▸), PLoS One, 9, e103470]. A number of additional proteins are thought to be involved in the formation of mRNP in yeast, including Tho1, which has been shown to bind RNA in vitro and is recruited to actively transcribed chromatin in vivo in a THO-complex and RNA-dependent manner. Tho1 is known to contain a SAP domain at the N-terminus, but the ability to suppress the expression defects of the hpr1Δ mutant of THO was shown to reside in the RNA-binding C-terminal region. In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and C-terminal RNA-binding domain have been determined

High-resolution NMR structures of the domains of Saccharomyces cerevisiae Tho1

Energy Technology Data Exchange (ETDEWEB)

Jacobsen, Julian O. B.; Allen, Mark D.; Freund, Stefan M. V.; Bycroft, Mark, E-mail: mxb@mrc-lmb.cam.ac.uk [MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH (United Kingdom)

2016-05-23

In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and the C-terminal RNA-binding domain of S. cerevisiae Tho1 have been determined. THO is a multi-protein complex involved in the formation of messenger ribonuclear particles (mRNPs) by coupling transcription with mRNA processing and export. THO is thought to be formed from five subunits, Tho2p, Hpr1p, Tex1p, Mft1p and Thp2p, and recent work has determined a low-resolution structure of the complex [Poulsen et al. (2014 ▸), PLoS One, 9, e103470]. A number of additional proteins are thought to be involved in the formation of mRNP in yeast, including Tho1, which has been shown to bind RNA in vitro and is recruited to actively transcribed chromatin in vivo in a THO-complex and RNA-dependent manner. Tho1 is known to contain a SAP domain at the N-terminus, but the ability to suppress the expression defects of the hpr1Δ mutant of THO was shown to reside in the RNA-binding C-terminal region. In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and C-terminal RNA-binding domain have been determined.

The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation.

Science.gov (United States)

Li, Ping; Stumpf, Maria; Müller, Rolf; Eichinger, Ludwig; Glöckner, Gernot; Noegel, Angelika A

2017-08-22

SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

Complex Correspondence Principle

International Nuclear Information System (INIS)

Bender, Carl M.; Meisinger, Peter N.; Hook, Daniel W.; Wang Qinghai

2010-01-01

Quantum mechanics and classical mechanics are distinctly different theories, but the correspondence principle states that quantum particles behave classically in the limit of high quantum number. In recent years much research has been done on extending both quantum and classical mechanics into the complex domain. These complex extensions continue to exhibit a correspondence, and this correspondence becomes more pronounced in the complex domain. The association between complex quantum mechanics and complex classical mechanics is subtle and demonstrating this relationship requires the use of asymptotics beyond all orders.

Gastric antral vascular ectasia and its clinical correlates in patients with early diffuse systemic sclerosis in the SCOT trial.

Science.gov (United States)

Hung, Emily W; Mayes, Maureen D; Sharif, Roozbeh; Assassi, Shervin; Machicao, Victor I; Hosing, Chitra; St Clair, E William; Furst, Daniel E; Khanna, Dinesh; Forman, Stephen; Mineishi, Shin; Phillips, Kristine; Seibold, James R; Bredeson, Christopher; Csuka, Mary Ellen; Nash, Richard A; Wener, Mark H; Simms, Robert; Ballen, Karen; Leclercq, Sharon; Storek, Jan; Goldmuntz, Ellen; Welch, Beverly; Keyes-Elstein, Lynette; Castina, Sharon; Crofford, Leslie J; Mcsweeney, Peter; Sullivan, Keith M

2013-04-01

To describe the prevalence and clinical correlates of endoscopic gastric antral vascular ectasia (GAVE; "watermelon stomach") in early diffuse systemic sclerosis (SSc). Subjects with early, diffuse SSc and evidence of specific internal organ involvement were considered for the Scleroderma: Cyclophosphamide Or Transplant (SCOT) trial. In the screening procedures, all patients underwent upper gastrointestinal endoscopy. Patients were then categorized into those with or without endoscopic evidence of GAVE. Demographic data, clinical disease characteristics, and autoantibody data were compared using Pearson chi-square or Student t tests. Twenty-three of 103 (22.3%) individuals were found to have GAVE on endoscopy. Although not statistically significant, anti-topoisomerase I (anti-Scl70) was detected less frequently among those with GAVE (18.8% vs 44.7%; p = 0.071). Similarly, anti-RNP antibodies (anti-U1 RNP) showed a trend to a negative association with GAVE (0 vs 18.4%; p = 0.066). There was no association between anti-RNA polymerase III and GAVE. Patients with GAVE had significantly more erythema or vascular ectasias in other parts of the stomach (26.1% vs 5.0%; p = 0.003). Endoscopic GAVE was present on screening in almost one-fourth of these highly selected patients with early and severe diffuse SSc. While anti-Scl70 and anti-U1 RNP trended toward a negative association with GAVE, there was no correlation between anti-RNA Pol III and GAVE. Patients with GAVE had a higher frequency of other gastric vascular ectasias outside the antrum, suggesting that GAVE may represent part of the spectrum of the vasculopathy in SSc.

Rheumatic Disease Autoantibodies in Autoimmune Liver Diseases.

Science.gov (United States)

Utiyama, Shirley R R; Zenatti, Katiane B; Nóbrega, Heloisa A J; Soares, Juliana Z C; Skare, Thelma L; Matsubara, Caroline; Muzzilo, Dominique A; Nisihara, Renato M

2016-08-01

Autoimmune liver diseases (ALDs) are known to be associated with systemic autoimmune rheumatic diseases (SARDs) and their autoantibodies. We aimed to study the prevalence of SARDs and related autoantibodies, as well as their prognostic implications in a group of patients with ALDs. This was a cross-sectional study. Sixty patients with ALDs (38.3% with autoimmune hepatitis; 11.7% with primary biliary cirrhosis; 25% with primary sclerosing cholangitis and 25% with overlap syndrome) were studied for the presence of SARDs and their autoantibodies. There was autoimmune rheumatic disease in 20% of the studied sample. Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were the commonest (11.6% and 5%, respectively). Antinuclear antibodies (ANAs) were present in 35% of the patients, followed by anti-Ro (20.0%); anti-nucleosome (18.3%); rheumatoid factor (10%) anti-CCP (8.3%); anti-RNP (8.3%); anti-ds-DNA (6.6%); anti-La (3.3%); anti-Sm (3.3%), anti-ribosomal P (3.3%). Anti-Ro (p = 0.0004), anti-La (p = 0.03), anti-RNP (p = 0.04) and anti-Sm (p = 0.03) were commonly found in patients with SARD, but not anti-DNA, anti-nucleosome and anti-ribosomal P. No differences were found in liver function tests regarding to the presence of autoantibodies. There was a high prevalence of SARD and their autoantibodies in ALD patients. Anti-Ro, anti-La, anti-RNP and anti-Sm positivity points to an association with systemic autoimmune rheumatic diseases. The presence of autoantibodies was not related to liver function tests.

Uranium thiolate complexes

International Nuclear Information System (INIS)

Leverd, Pascal C.

1994-01-01

This research thesis proposes a new approach to the chemistry of uranium thiolate complexes as these compounds are very promising for various uses (in bio-inorganic chemistry, in some industrial processes like oil desulphurization). It more particularly addresses the U-S bond or more generally bonds between polarizable materials and hard metals. The author thus reports the study of uranium organometallic thiolates (tricyclo-penta-dienic and mono-cyclo-octa-tetraenylic complexes), and of uranium homoleptic thiolates (tetra-thiolate complexes, hexa-thiolate complexes, reactivity of homoleptic thiolate complexes) [fr

Clinical Complexity in Medicine: A Measurement Model of Task and Patient Complexity.

Science.gov (United States)

Islam, R; Weir, C; Del Fiol, G

2016-01-01

Complexity in medicine needs to be reduced to simple components in a way that is comprehensible to researchers and clinicians. Few studies in the current literature propose a measurement model that addresses both task and patient complexity in medicine. The objective of this paper is to develop an integrated approach to understand and measure clinical complexity by incorporating both task and patient complexity components focusing on the infectious disease domain. The measurement model was adapted and modified for the healthcare domain. Three clinical infectious disease teams were observed, audio-recorded and transcribed. Each team included an infectious diseases expert, one infectious diseases fellow, one physician assistant and one pharmacy resident fellow. The transcripts were parsed and the authors independently coded complexity attributes. This baseline measurement model of clinical complexity was modified in an initial set of coding processes and further validated in a consensus-based iterative process that included several meetings and email discussions by three clinical experts from diverse backgrounds from the Department of Biomedical Informatics at the University of Utah. Inter-rater reliability was calculated using Cohen's kappa. The proposed clinical complexity model consists of two separate components. The first is a clinical task complexity model with 13 clinical complexity-contributing factors and 7 dimensions. The second is the patient complexity model with 11 complexity-contributing factors and 5 dimensions. The measurement model for complexity encompassing both task and patient complexity will be a valuable resource for future researchers and industry to measure and understand complexity in healthcare.

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster.

Directory of Open Access Journals (Sweden)

Patricia L Graham

2011-07-01

Full Text Available In female fruit flies, Sex-lethal (Sxl turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2 mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf, the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2d--that have been directly implicated in Sxl splicing regulation.

Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide

Directory of Open Access Journals (Sweden)

Hinnebusch Alan G

2011-01-01

Full Text Available Abstract Background Eukaryotic translation initiation factor 4G (eIF4G is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs, using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes. Results Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5 on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes. Conclusions This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m7 G cap- and poly(A-binding factors (eIF4E and PABP, is not essential for translation of most (if not all mRNAs but enhances the differentiation of translational efficiencies genome-wide.

Thinking Forbidden Thoughts: The Oedipus Complex as a Complex of Knowing.

Science.gov (United States)

Schein, Michael

2016-04-01

The Oedipus complex, considered by Freud the "nuclear complex of development," played a central role in the evolution of psychoanalytic thought. This paper returns to the point of transition from the seduction theory, Freud's initial theorem, to the oedipal model, and suggests that the Oedipus complex is first and foremost a text and as such contains a multiplicity of narratives. In particular, the author articulates the close relation between the Oedipus complex and the subject of knowing, postulating that underlying its surface level, the deep-level structure of this complex is one of knowing. As a complex of knowing it is of dual quality, both promoting and impeding the ability to know.

Characterization of MRP RNA–protein interactions within the perinucleolar compartment

Science.gov (United States)

Pollock, Callie; Daily, Kelly; Nguyen, Van Trung; Wang, Chen; Lewandowska, Marzena Anna; Bensaude, Olivier; Huang, Sui

2011-01-01

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA–processing (MRP) RNA, pyrimidine tract–binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA–containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)–PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA–protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC. PMID:21233287

Complexity-management in SME : organization of complex relationships

NARCIS (Netherlands)

Gregus, M.; Mandorf, S.

2009-01-01

The complexity of companies' environment IS growmg. Complexity management and restructuring of small and medium-sized enterprises (SME) become big challenges of business studies in the next future. A chance could be seen in the use of e-business strategies and the implementation of information

Managing Complexity

DEFF Research Database (Denmark)

Maylath, Bruce; Vandepitte, Sonia; Minacori, Patricia

2013-01-01

and into French. The complexity of the undertaking proved to be a central element in the students' learning, as the collaboration closely resembles the complexity of international documentation workplaces of language service providers. © Association of Teachers of Technical Writing.......This article discusses the largest and most complex international learning-by-doing project to date- a project involving translation from Danish and Dutch into English and editing into American English alongside a project involving writing, usability testing, and translation from English into Dutch...

Mathematics for electric engineers. Complex numbers; Mathematiques pour l`electricien. Nombres complexes

Energy Technology Data Exchange (ETDEWEB)

Rouxel, C. [Conservatoire National des Arts et Metiers (CNAM), 75 - Paris (France)

1999-05-01

Complex numbers are widely used in electrical engineering. This article is divided into 5 parts dealing successively with: the cartesian form of complex numbers (definition, conjugated complex numbers, graphical representation); the trigonometrical form of complex numbers (module and argument, trigonometrical form, exponential notation, multiplication and division of two complex numbers); Moivre and Euler formulae; applications (square root and second degree equation, n. roots, plan rotation and similarity); cissoidal transformation (definition, properties, applications to electricity: complex impedance in permanent sinusoidal regime, transfer function of a linear system in permanent regime, study of an example). (J.S.)

Identification of Methylosome Components as Negative Regulators of Plant Immunity Using Chemical Genetics.

Science.gov (United States)

Huang, Shuai; Balgi, Aruna; Pan, Yaping; Li, Meng; Zhang, Xiaoran; Du, Lilin; Zhou, Ming; Roberge, Michel; Li, Xin

2016-12-05

Nucleotide-binding leucine-rich repeat (NLR) proteins serve as immune receptors in both plants and animals. To identify components required for NLR-mediated immunity, we designed and carried out a chemical genetics screen to search for small molecules that can alter immune responses in Arabidopsis thaliana. From 13 600 compounds, we identified Ro 8-4304 that was able to specifically suppress the severe autoimmune phenotypes of chs3-2D (chilling sensitive 3, 2D), including the arrested growth morphology and heightened PR (Pathogenesis Related) gene expression. Further, six Ro 8-4304 insensitive mutants were uncovered from the Ro 8-4304-insensitive mutant (rim) screen using a mutagenized chs3-2D population. Positional cloning revealed that rim1 encodes an allele of AtICln (I, currents; Cl, chloride; n, nucleotide). Genetic and biochemical analysis demonstrated that AtICln is in the same protein complex with the methylosome components small nuclear ribonucleoprotein D3b (SmD3b) and protein arginine methyltransferase 5 (PRMT5), which are required for the biogenesis of small nuclear ribonucleoproteins (snRNPs) involved in mRNA splicing. Double mutant analysis revealed that SmD3b is also involved in the sensitivity to Ro 8-4304, and the prmt5-1 chs3-2D double mutant is lethal. Loss of AtICln, SmD3b, or PRMT5 function results in enhanced disease resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, suggesting that mRNA splicing plays a previously unknown negative role in plant immunity. The successful implementation of a high-throughput chemical genetic screen and the identification of a small-molecule compound affecting plant immunity indicate that chemical genetics is a powerful tool to study whole-organism plant defense pathways. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

ComplexRec 2017

DEFF Research Database (Denmark)

a single step in the user's more complex background need. These background needs can often place a variety of constraints on which recommendations are interesting to the user and when they are appropriate. However, relatively little research has been done on these complex recommendation scenarios....... The ComplexRec 2017 workshop addressed this by providing an interactive venue for discussing approaches to recommendation in complex scenarios that have no simple one-size-fits-all-solution....

Complex variables

CERN Document Server

Fisher, Stephen D

1999-01-01

The most important topics in the theory and application of complex variables receive a thorough, coherent treatment in this introductory text. Intended for undergraduates or graduate students in science, mathematics, and engineering, this volume features hundreds of solved examples, exercises, and applications designed to foster a complete understanding of complex variables as well as an appreciation of their mathematical beauty and elegance. Prerequisites are minimal; a three-semester course in calculus will suffice to prepare students for discussions of these topics: the complex plane, basic

Complexity Management In Projects Between Rational Momentum And Complex Conditions

DEFF Research Database (Denmark)

Mac, Anita; Schlamovitz, Jesper

2015-01-01

Abstract: This study takes its departure in a model of complexity, developed by Stacey (1993), to test and discuss its practical benefit as perceived by practicing project managers. Based on a survey, the study finds that complexity is a phenomenon recognized by project managers, and complexity...... management is associated with benefits in the development of tasks and managing stakeholders. It is also associated with some difficulty in terms of an increased need for dialogue and a risk of creating goal ambiguity. Based on the findings, we conclude that classical project management approaches can...... benefit from incorporating complexity management....

Complexity management in projects between rational momentum and complex conditions

DEFF Research Database (Denmark)

Mac, Anita; Schlamovitz, Jesper

This study takes its departure in a model of complexity, developed by Stacey (1993), to test and discuss its practical benefit as perceived by practicing project managers. Based on a survey, the study finds that complexity is a phenomenon recognized by project managers, and complexity management...... is associated with benefits in the development of tasks and managing stakeholders. It is also associated with some difficulty in terms of an increased need for dialogue and a risk of creating goal ambiguity. Based on the findings, we conclude that classical project management approaches can benefit from...... incorporating complexity management....

RNA recognition motif (RRM)-containing proteins in Bombyx mori

African Journals Online (AJOL)

STORAGESEVER

2009-03-20

Mar 20, 2009 ... Recognition Motif (RRM), sometimes referred to as. RNP1, is one of the first identified domains for RNA interaction. RRM is very common ..... Apart from the RRM motif, eIF3-S9 has a Trp-Asp. (WD) repeat domain, Poly (A) ...

Sklerodermi--systemisk sklerose. Serologi, lungefunktion og overlevelse

DEFF Research Database (Denmark)

Ullman, S; Halberg, P; Wiik, A

1999-01-01

%, anti-Scl-70 antibodies in 13% and anti-U1-RNP antibodies in 6.5%. These serological groups were associated with limited SSc, diffuse SSc, and myositis/arthritis, respectively. The most prevalent finding at first lung function test was isolated reduction of diffusion capacity (47%). Further...

Transmission and pathogenesis of vesicular stomatitis viruses

Science.gov (United States)

Vesicular Stomatitis (VS) is caused by the Vesicular Stomatitis Virus (VSV), a negative single stranded RNA arthropod-borne virus member of the Family Rhabdoviridae. The virion is composed of the host derived plasma membrane, the envelope, and an internal ribonucleoprotein core. The envelope contain...

Sequence Classification: 893543 [

Lifescience Database Archive (English)

Full Text Available rich protein with a role in preribosome assembly or transport; may function as a chaperone of small nucleolar ribonucleoprotein parti...cles (snoRNPs); immunologically and structurally to rat Nopp140; Srp40p || http://www.ncbi.nlm.nih.gov/protein/6322945 ...

Predictors of intestinal pseudo-obstruction in systemic lupus erythematosus complicated by digestive manifestations: data from a Southern China lupus cohort.

Science.gov (United States)

Huang, Q; Lai, W; Yuan, C; Shen, S; Cui, D; Zhao, J; Lin, J; Ren, H; Yang, M

2016-03-01

To determine factors that may predict intestinal pseudo-obstruction (IpsO) in systemic lupus erythematosus (SLE) patients complicated by digestive manifestations. SLE patients with digestive manifestations (n = 135) were followed at Southern Medical University affiliated Nanfang Hospital from 2000 until 2013. Demographic variables, clinical features, and laboratory data were compared between the two groups. Univariate and multivariate logistic regression models were used to establish factors that predispose to IpsO in these patients. At the end of the study period, 32 (23.7%) patients had developed IpsO. Mortality (9 patients) was infrequent and the cause of death was unrelated to IpsO. Independent predictors of IpsO in SLE were ureterectasia, anti-U1 RNP(+), peritonitis, and low C3 levels. Regular abdominal X-ray examinations are recommended in SLE patients with ureterectasia, anti-U1 RNP(+), peritonitis, or low C3 levels, as early diagnosis and therapy may prevent unnecessary surgical intervention and improve the disease course. © The Author(s) 2015.

Significance of combined determination of multiple autoantibodies in patients with systemic lupus erythematosus

International Nuclear Information System (INIS)

Tao Hongqun; Li Xiaolong; Gong Jianguang; Wen Huaikai

2006-01-01

Objective: To explore the roles played by autoantibodies in systemic lupus erythematosus. Methods: Serum anti-dsDNA antibody (with RIA) and serum anti-nucleosome antibody (AnuA), AHA, anti SmD1-, anti Ro60UD-, anti U1 -RNP- , anti-Ro52KD, anti-SSB antibodies (with anti-nucleo antibodies linear spectrum blotting method) were detected in 50 patients with clinically proven systemic lupus erythematosus. Results: The positive rate with anti-SmD1 antibody was highest (82%), followed by anti-Ro60KD antibody (80%) and AnuA (72%). Positive rate with anti dsDNA-, AHA, anti-U1-RNP-, anti- Ro52KD and anti SSB-antibodies was 44%, 32%, 58%, 48% and 24% respectively. Positive rate with anti-SC1-70, ACA and Jo-1 antibodies was extremely low (below 10%). Conclusion: Multiple auto-antibodies were present in serum of patients with systemic lupus erythematosus and combined detection of them would improve the diagnostic sensitivity. (authors)

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

Science.gov (United States)

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

Directory of Open Access Journals (Sweden)

Joanna J Moser

2010-10-01

Full Text Available GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.