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At least 253 records · Page 14Linked to original sources

PubMed2006

Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules.

Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function.

APOBEC-3G Deaminase↗

PubMed1981

Cytoplasmic nonpolysomal ribonucleoprotein complexes and translational control.

In this article, we discuss our attempts to establish the existence in the cytoplasm of regulatory molecules involved in translational control. Our studies have revealed the presence of cAMP independent protein kinase in the free mRNP complex capable of phosphorylating a Mr = 38 000 polypeptide, also part of the same complex. Both the kinase and the acceptor protein were found also as free proteins in the cytoplasmic pool. This kinase has been shown to be distinct from the heme regulated enzyme that phosphorylates the small subunit of eIF-2. Other regulatory molecules include small molecular weight RNAs found as part of an RNP complex. A 4S fraction isolated from this complex inhibited the translation of both capped and uncapped mRNAs in a cell-free protein synthesizing system. The biological role of the protein kinase and the 4S RNA fraction is considered.

Animals↗

PubMed2006

Influenza virus pleiomorphy characterized by cryoelectron tomography.

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles. In this study, we used cryoelectron tomography to visualize the 3D structures of 110 individual virions of the X-31 (H3N2) strain of influenza A. The tomograms distinguish two kinds of glycoprotein spikes [hemagglutinin (HA) and neuraminidase (NA)] in the viral envelope, resolve the matrix protein layer lining the envelope, and depict internal configurations of ribonucleoprotein (RNP) complexes. They also reveal the stems that link the glycoprotein ectodomains to the membrane and interactions among the glycoproteins, the matrix, and the RNPs that presumably control the budding of nascent virions from host cells. Five classes of virions, four spherical and one elongated, are distinguished by features of their matrix layer and RNP organization. Some virions have substantial gaps in their matrix layer ("molecular fontanels"), and others appear to lack a matrix layer entirely, suggesting the existence of an alternative budding pathway in which matrix protein is minimally involved.

Animals↗

PubMed1986

U1, U2, and U6 small nuclear ribonucleoproteins (snRNPs) are associated with large nuclear RNP particles containing transcripts of an amplified gene in vivo.

Nuclear ribonucleoprotein (RNP) complexes that contain intact transcripts of the amplified gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells, have been released from nuclei of Syrian hamster cells as large particulate structures that sediment at the 200S region in a sucrose gradient. By the technique of RNA hybridization, we have shown that U1, U2, and U6 small nuclear RNAs (snRNAs) cosediment with the large RNP particles in the sucrose gradients. Autoimmune sera from systemic lupus erythematosus and mixed connective tissue disease patients, characterized as anti-(U1)RNP, have further been shown to immunoprecipitate CAD RNA along with U1 and U2 snRNAs from the fractionated nuclear 200S RNP particles. We conclude that U1, U2, and U6 snRNPs are integral constituents of the 200S RNP particles. The requirement of snRNPs for RNA processing that evidently occurs on RNP particles has been recently demonstrated. Our results thus suggest that the 200S RNPs are structurally and functionally close to the native particles on which RNA processing occurs.

Aspartate Carbamoyltransferase↗

PubMed1992

Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes.

A new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone. Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the full-length negative-strand RNA of the CAT gene positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain. When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexamethasone.

Animals↗

PubMed1999

Phosphorylation of tobacco mosaic virus movement protein abolishes its translation repressing ability.

Previously we showed that the ribonucleoprotein complexes (RNPs) of the TMV 30-kDa movement protein (MP) with TMV RNA are nontranslatable in vitro and noninfectious to protoplasts, but are infectious to intact plants. It has been suggested that MP-TMV RNA complexes could be converted into the translatable and replicatable form in planta in the course of passage through plasmodesmata (Karpova et al., 1997, Virology 230, 11-21). The role of TMV MP phosphorylation was investigated in terms of its capacity to modulate the translation-repressing ability of the MP. Phosphorylation of the TMV MP, either before or after RNP complex formation, caused a conversion of nontranslatable MP-RNA complexes into a form that was translatable in vitro and infectious to protoplasts and plants.

Brassica↗

PubMed2006

Membrane accumulation of influenza A virus hemagglutinin triggers nuclear export of the viral genome via protein kinase Calpha-mediated activation of ERK signaling.

Replication and transcription of the influenza virus genome takes place exclusively within the nucleus of the infected cells. The viral RNA genome, polymerase subunits, and nucleoprotein form ribonucleoprotein (RNP) complexes. Late in the infectious cycle RNPs have to be exported from the nucleus to be enwrapped into budding progeny virions at the cell membrane. This process requires viral activation of the cellular Raf/MEK/ERK (mitogen-activated protein kinase (MAPK)) signaling cascade that is activated late in the infection cycle. Accordingly, block of the cascade results in retardation of RNP export and reduced titers of progeny virus. In the present study we have analyzed the importance of cell-membrane association of the viral hemagglutinin glycoprotein for viral MAPK activation. We show that hemagglutinin membrane accumulation and its tight association with lipid-raft domains trigger activation of the MAPK cascade via protein kinase Calpha activation and induces RNP export. This may represent an auto-regulative mechanism that coordinates timing of RNP export to a point when all viral components are ready for virus budding.

Animals↗

PubMed1984

Polysomal and cytoplasmic mRNP particles containing 7S(L) RNA.

Nuclear RNP complexes, cytoplasmic mRNP particles and free and membrane-bound polysomes were prepared from rat liver and their low-molecular-mass RNA components were analyzed on polyacrylamide/formamide gels. The separated small RNAs transferred to diazophenylthioether paper were hybridized to the nick-translated recombinant plasmid pA6 containing cDNA sequences for the low-Mr RNA called 7S(L) RNA. Nuclear RNP particles and free and membrane-bound polysomes were found to contain 7S(L) RNA. In the cytoplasm 7S(L) RNA could be identified as the major small RNA in 20-S cmRNP particles.

Animals↗

PubMed1989

The ribonucleoprotein structures along the pathway of mRNA formation.

Heterogeneous nuclear RNAs (hnRNAs), some of which are mRNA precursors, and the mature mRNAs are associated in eukaryotic cells with specific proteins to form ribonucleoprotein complexes (RNP). The RNP proteins are likely to play a major role in the formation, packaging, processing, and function of mRNA. The major proteins that interact with hnRNA and with mRNA were identified by photochemical RNA-protein cross-linking in intact cells and monoclonal antibodies to several of these proteins were produced. Using these antibodies the hnRNP proteins were characterized and the hnRNP complex was isolated from vertebrate cell nuclei. The hnRNP complex is a unitary structure of consistent, defined and conserved components. The proteins of the hnRNP complex were identified and the general organization of hnRNA and proteins in the hRNP complex were studied.

Animals↗

PubMed1981

Structural analysis of hnRNP particles approached by in vitro phosphorylation using exogenous protein kinase and l gamma 32 P1 ATP.

Using an exogenous kinase, nuclear ribonucleoprotein complexes with sedimentation coefficients greater than 100S were phosphorylated in vitro before and after treatment with increasing concentrations of NaC1. The phosphorylation pattern of the proteins before raising the NaC1 concentration shows a major group of labelled proteins in the 30 000 to 40 000 MW range. Treatment of the complexes with 400 and 800 mM NaC1 produces a relative increase in the labelling of some polypeptides with the appearance of new labelled bands and the concomitant disappearance of several proteins. Even at the highest salt concentration used (1.2 M), it is still possible to identify a group of labelled polypeptides which are suggested to form the backbone structure of the nuclear RNP complexes.

Adenosine Triphosphate↗

PubMed2002

The product of the survival of motor neuron (SMN) gene is a human telomerase-associated protein.

Telomerase is a ribonucleoprotein (RNP) complex that is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA component, the telomerase RNA. The survival of motor neuron (SMN) gene codes for a protein involved in the biogenesis of certain RNPs. Here, we report that SMN is a telomerase-associated protein. Using in vitro binding assays and immunoprecipitation experiments, we demonstrate an association between SMN and the telomerase RNP in vitro and in human cells. The specific immunopurification of SMN from human 293 cells copurified telomerase activity, suggesting that SMN associates with a subset of the functional telomerase holoenzyme. Our results also indicate that the human telomerase RNA and the human (h) TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase. Immunofluorescence analysis showed that hTERT does not specifically colocalize with wild-type SMN in gems or Cajal bodies. However, a dominant-negative mutant of SMN (SMNDeltaN27) previously characterized to elicit the cellular reorganization of small nuclear RNPs caused the accumulation of hTERT in specific SMNDeltaN27-induced cellular bodies. Furthermore, coexpression of SMNDeltaN27 and hTERT in rabbit reticulocyte lysates decreased the efficiency of human telomerase reconstitution in vitro. Our results establish SMN as a novel telomerase-associated protein that is likely to function in human telomerase biogenesis.

Catalysis↗

PubMed1981

Analysis of reconstruction of an RNP particle which stores 5S RNA and tRNA in amphibian oocytes.

Previtellogenic oocytes of Triturus cristatus accumulate a free cytoplasmic RNP which sediments at 40S and contain 5S RNA and tRNA in association with two proteins of MW 45,000 and 39,000 daltons (P45 and P39). The 40S particle has a buoyant density of 1.53 g . cm-3 in CsCl and consists of four identical RNP subunits. Each monomeric subunit contains one molecule of 5S RNA, three molecules of tRNA, two molecules of P45 and one molecule of P39. The 40S particle can be completely dissociated by SDS treatment into its individual components, and the subunits, and even the complete 40S particle, can be reformed by removal of SDS in the presence of 0.2 M NaCl. RNA/protein binding experiments with isolated components, and analysis of reformed RNP complexes in CsCl gradients, demonstrate that the stable interactions are: 5S RNA/P45, 3(tRNA)/P45, 5S RNA/P39 and 5S RNA/P45/P39. Immunological studies show that P45 has also a nuclear location and may bind to the 5S RNA transcript in the chromatin, whereas P39 is predominantly cytoplasmic and is possibly related to proteins associated with 5S RNA in the ribosomal 60S subunit. It is suggested that the 40S RNP particle not only stores 5S RNA and tRNA but also provides a means for the exchange of the 5S RNA transcript binding protein (P45) for the 5S RNA ribosome associated protein (P39).

Animals↗

PubMed1999

Characterization of the 5' ends for polyadenylated RNAs synthesized during the replication of hepatitis delta virus.

The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5' rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5' ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5' ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5' ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5' ends. The observed end located at nt 1630, and most of the novel 5' ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5' ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5' ends could be controlled by nucleotide sequences at and around nt 1630.

5' Untranslated Regions↗

PubMed2003

Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E.

Influenza virus polymerase uses capped RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonuclease at a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excess uncapped RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involve synergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode of interaction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of 7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contribution from the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction, it was possible to identify novel cap analogues, which selectively interact with influenza virus, but not human cap binding protein.

Base Sequence↗

PubMed1988

trans splicing in Leishmania enriettii and identification of ribonucleoprotein complexes containing the spliced leader and U2 equivalent RNAs.

The 5' ends of Leishmania mRNAs contain an identical 35-nucleotide sequence termed the spliced leader (SL) or 5' mini-exon. The SL sequence is at the 5' end of an 85-nucleotide primary transcript that contains a consensus eucaryotic 5' intron-exon splice junction immediately 3' to the SL. The SL is added to protein-coding genes immediately 3' to a consensus eucaryotic 3' intron-exon splice junction. Our previous work demonstrated possible intermediates in discontinuous mRNA processing that contain the 50 nucleotides of the SL primary transcript 3' to the SL, the SL intron sequence (SLIS). These RNAs have a 5' terminus at the splice junction of the SL and the SLIS. We examined a Leishmania nuclear extract for these RNAs in ribonucleoprotein (RNP) particles. Density centrifugation analysis showed that the SL RNA is predominantly in RNP complexes at 60S, while the SLIS-containing RNAs are in complexes at 40S. We also demonstrated that the SLIS can be released from polyadenylated RNA by incubation with a HeLa cell extract containing debranching enzymatic activity. These data suggested that Leishmania enriettii mRNAs are assembled by bimolecular or trans splicing as has been recently demonstrated for Trypanosoma brucei. Furthermore, we determined the partial sequence of the Leishmania U2 equivalent RNA and demonstrated that it cosediments with the SL RNA at 60S in a nuclear extract. These RNP particles may be analogous to so-called spliceosomes that have been demonstrated in other systems.

Animals↗

PubMed2000

Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1.

For nuclear export of proteins, the formation of a ternary export complex composed of the export substrate, a cellular export factor and Ran-GTP is crucial. CRM1 is a cellular export factor for proteins containing leucine-rich nuclear export signals (NESs). Although the NES sequence is crucial for nuclear export, its exact role in the formation of the ternary export complex is controversial. Here we demonstrate an interaction between human CRM1 (hCRM1) and influenza A virus NS2 protein, which contains an NES motif in its N-terminal region. Replacement of the hydrophobic amino acids in the NES motif did not abolish NS2's interaction with hCRM1. Using our recently established systems for the generation of influenza virus or virus-like particles from cloned cDNAs, we found that NS2 is essential for nuclear export of influenza virus ribonucleoprotein (RNP) complexes, and that alteration of the NS2-NES abrogated this event and influenza virus generation. These findings suggest that the NS2-NES is not crucial for the interaction of this protein with hCRM1, but is for the formation of the ternary export complex with Ran-GTP.

Active Transport, Cell Nucleus↗

PubMed2001

Cloning and characterization of two guide RNA-binding proteins from mitochondria of Crithidia fasciculata: gBP27, a novel protein, and gBP29, the orthologue of Trypanosoma brucei gBP21.

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.

Amino Acid Sequence↗

PubMed2003

Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA. Crystal structure of an oligomeric complex of yeast SmF.

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.

Binding Sites↗