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PubMed1991

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

Animals↗
PubMed2001

Human telomerase RNA-protein interactions.

Telomere length is maintained in most eukaryotic cells by telomerase. The core components of this ribonucleoprotein (RNP) enzyme include a protein catalytic subunit, composed of motifs conserved among reverse transcriptases (RT), and an RNA subunit that contains a short template sequence essential for the synthesis of telomeric repeats. We developed an electrophoretic mobility shift assay using active telomerase partially purified from 293 cells and radiolabeled, in vitro-transcribed human telomerase RNA (hTR) to investigate the molecular interactions of the human telomerase RT (hTERT) and telomerase-associated proteins with hTR. A specific hTR-protein complex was identified and shown to contain hTERT and human Staufen by antibody supershift assays. Variants of hTR altered in distinct structural elements were analyzed for their ability to competitively inhibit complex formation. Human telomerase RNAs lacking the CR4-CR5 domain were poor inhibitors of hTR-protein complex formation, suggesting that the CR4-CR5 domain of hTR is a potential protein-binding site. Furthermore, alterations in the telomerase RNA pseudoknot's P3 helix, the CR7 domain, or the H/ACA box efficiently inhibited formation of the complex, indicating that these domains are dispensable for the assembly of a telomerase RNP in vitro. Potential telomerase-associated proteins that bind hTR were also identified using a UV cross-linking assay.

Binding Sites↗
PubMed1981

Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus.

The RNP and Sm antigens recognized by lupus erythematosus antibodies are located on discrete particles containing single small nuclear RNA's complexed with proteins. The antigens Ro and La are also on ribonucleoproteins. The small RNA's in ribonucleoproteins with Ro are discrete, like those associated with RNP and Sm; in contrast, ribonucleoproteins with La contain a striking highly banded spectrum of small RNA's from uninfected cells as well as virus-associated RNA from adenovirus-infected cells.

Animals↗
PubMed1996

RNP export is mediated by structural reorganization of the nuclear pore basket.

Messenger RNA leaves the cell nucleus as ribonucleoprotein (RNP) particles. The nucleocytoplasmic translocation of the particles takes place through the nuclear pore complex (NPC) and includes two steps: binding to the NPC and transit through its central channel. The NPC basket is a fishtrap-like component of NPC facing the nucleoplasm. Its position in the NPC strongly suggests that it has an important role in the initial steps of macromolecular export from the nucleus. Here we report a cyclic rearrangement of the basket structure in relation to the translocation of a specific messenger RNP (mRNP) of exceptional size, the Balbiani ring RNP particles in the salivary gland cells in Chironomus. We used field emission in-lens scanning electron microscopy (FEISEM), transmission electron microscopy (TEM), and immunocytochemistry to analyse the structural organization of the basket during the mRNP export. Our observations reveal five configurations of the basket which are presented in a model of basket reorganization related to the state of mRNP penetration into the NPC. We suggest that the functional role of the basket is to anchor the mRNP particle to the NPC and position it in correct orientation at the entrance to the central channel of the NPC.

Animals↗
PubMed2001

The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle.

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.

Animals↗
PubMed1979

Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus.

Patients with systemic lupus erythematosus often possess antibodies against two nuclear antigens called Sm and RNP (ribonucleoprotein). We have established the molecular identity of these antigens by analyzing immune precipitates of nuclear extracts from mouse Ehrlich ascites cells labeled with (32)P and (35)S. Anti-Sm serum selectively precipitates six small nuclear RNA molecules (snRNAs); anti-RNP serum reacts with only two of these; and a third serum, characterized as mostly anti-RNP, precipitates a subset of three snRNA bands. Three of the six RNAs are identified by fingerprint analysis as the previously characterized and highly abundant nucleoplasmic snRNA species U1a (171 nucleotides), U1b, and U2 (196 nucleotides). The other three RNAs (U4, U5, and U6) likewise are uridine rich and contain modified nucleotides, but they are smaller, with lengths of about 145, 120, and 95 residues, respectively. Each of the six snRNAs is complexed with and apparently antigenic by virtue of association with specific proteins. All three sera precipitate an identical complement of seven different polypeptides ranging in molecular weight from 12,000 to 35,000; these proteins are abundant in nuclear extracts, but are neither histones nor the major polypeptides comprising the 30S heterogeneous nuclear RNP particles of mammalian nuclei. Our data argue that each of the six snRNAs exists in a separate small nuclear ribonucleoprotein (snRNP) complex with a total molecular weight of about 175,000. We find that human antisera also precipitate snRNAs from a wide range of vertebrate species and from arthropods. We discuss the antigenic snRNPs in relation to the published literature on snRNAs and nuclear RNPs and consider possible functions of snRNPs in nuclear processes.

Antibodies, Antinuclear↗
PubMed1990

RNP, Sm and SS-B antigens from calf thymus: molecular stability upon enzymatic digestion.

RNP, Sm and SS-B nuclear antigens from calf thymus were studied with respect to the size distribution on sucrose gradients as well as to the molecular integrity and related structural changes when they were subjected to enzymatic digestions under different conditions. Making a difference with RNP particles, the Sm size distribution is concentration dependent, a property in accordance with the complexity of the Sm particles in comparison with the RNPs. The use of combined effects of temperature, endogenous proteases and RNase A, allowed us to gain insight into the limits of stability of the three antigenic particles. Following treatments in the absence of RNAse A, the degradation products (32-38 Kd molecular weight) of the 70 Kd RNP polypeptide remain stable and associated with other molecules within the RNP particle. It was also found that the phosphate groups of the SS-B protein moiety are only accessible to alkaline phosphatase if the RNA of the SS-B particle is degraded by the action of RNAse A.

Animals↗
PubMed1998

The hrp23 protein in the balbiani ring pre-mRNP particles is released just before or at the binding of the particles to the nuclear pore complex.

Balbiani ring (BR) pre-mRNP particles reside in the nuclei of salivary glands of the dipteran Chironomus tentans and carry the message for giant-sized salivary proteins. In the present study, we identify and characterize a new protein component in the BR ribonucleoprotein (RNP) particles, designated hrp23. The protein with a molecular mass of 20 kD has a single RNA-binding domain and a glycine-arginine-serine-rich auxiliary domain. As shown by immunoelectron microscopy, the hrp23 protein is added to the BR transcript concomitant with transcription, is still present in the BR particles in the nucleoplasm, but is absent from the BR particles that are bound to the nuclear pore complex or are translocating through the central channel of the complex. Thus, hrp23 is released just before or at the binding of the particles to the nuclear pore complex. It is noted that hrp23 behaves differently from two other BR RNP proteins earlier studied: hrp36 and hrp45. These proteins both reach the nuclear pore complex, and hrp36 even accompanies the RNA into the cytoplasm. It is concluded that each BR RNA-binding protein seems to have a specific flow pattern, probably related to the particular role of the protein in gene expression.

Amino Acid Sequence↗
PubMed2004

RNA-guided nucleotide modification of ribosomal and non-ribosomal RNAs in Archaea.

Archaea use ribonucleoprotein (RNP) machines similar to those found in the eukaryotic nucleolus to methylate ribose residues in nascent ribosomal RNA. The archaeal complex required for this 2'-O-ribose-methylation consists of the C/D box sRNA guide and three proteins, the core RNA-binding aL7a protein, the aNop56 protein and the methyltransferase aFib protein. These RNP machines were reconstituted in vitro from purified recombinant components, and shown to have methylation activity when provided with a simple target oligonucleotide, complementary to the sRNA guide sequence. To obtain a better understanding of the versatility and specificity of this reaction, the activity of reconstituted particles on more complex target substrates, including 5S RNA, tRNA(Gln) and 'double target' oligonucleotides that exhibit either direct or reverse complementarity to both the D' and D box guides, has been examined. The natural 5S and tRNA(Gln) substrates were efficiently methylated in vitro, as long as the complementarity between guide and target was about 10 base pairs in length, and lacked mismatches. Maximal activity of double guide sRNAs required that both methylation sites be present in cis on the target RNA.

Archaeal Proteins↗
PubMed2003

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy.

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.

Animals↗
PubMed1981

Patterns of transcriptional activity of nucleolar genes during progesterone-induced maturation of oocytes of Xenopus laevis.

Maturation has been induced in full grown oocytes of Xenopus laevis by incubation in progesterone, and the ultrastructure of their nucleoli has been examined by electron microscopy using the chromatin spreading technique. We show that in this species numerous extrachromosomal nucleoli maintain high levels of transcription of rDNA for up to 200 min after the application of the hormone, i.e., shortly before germinal vesicle breakdown. Transcription has been identified as normal arrays of matrix units containing densely packed transcriptional complexes. In addition to normal-sized arrays of gradients of nascent RNP fibrils, as typical of active pre-rRNA genes, a number of unusual structures are described which include situations of sparse coverage of lateral fibrils in some matrix units, indicative of reduced frequencies of initiation events. The observations are discussed in relation to the time course of nucleolar gene inactivation described in oocytes of other amphibian species, as well as in relation to inactivation of chromosomal genes characteristic of this step of meiotic prophase I.

Animals↗
PubMed2000

The Ct-RAE1 protein interacts with Balbiani ring RNP particles at the nuclear pore.

RAE1 is an evolutionarily conserved protein that associates with both mRNPs and nucleoporins, and may bridge the interaction between mRNP export cargoes and the nuclear pore complex (NPC). However, the mechanism by which RAE1 functions in mRNA export is still unknown and the time point at which RAE1 interacts with the exported RNP has not been directly investigated. Here we have addressed this question in the Balbiani ring (BR) system of Chironomus tentans using immunoelectron microscopy. The RAE1 protein of C. tentans, Ct-RAE1, is 70% identical to human RAE1/mrnp41 (hRAE1) and is recognized by antibodies raised against hRAE1. As in vertebrate cells, Ct-RAE1 is concentrated at the nuclear envelope and also dispersed throughout the nuclear interior. Here we show that Ct-RAE1 does not bind to the BR particle either cotranscriptionally or in the nucleoplasm. Instead, the interaction between Ct-RAE1 and the exported BR particle occurs at the NPC. Moreover, the localization of Ct-RAE1 at the NPC is correlated with the presence of an exported RNP in the NPC. Finally, the anti-RAE1 antibody does not label the cytoplasmic side of BR particles in transit through the central channel, which indicates that Ct-RAE1 either remains anchored at the nuclear side of the NPC during translocation of the RNP through the central channel or becomes transiently associated with the RNP but is rapidly released into the cytoplasm.

Amino Acid Sequence↗
PubMed1990

Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. II. Protection from influenza infection and mechanism of protection.

Protection against influenza A virus infection in mice immunized with recombinant nucleoprotein (rNP) was studied. Nucleoprotein-immune mice were protected against a lethal challenge with A/Puerto Rico/8/34 (A/PR8) virus but showed considerable morbidity before recovery. Local boosting of the immune system with rNP by intranasal immunization improved the protection in NP-immune mice, and the decrease in morbidity after infection was reflected in accelerated clearance of virus from lungs. However, immune, boosted mice also rapidly cleared an antigenically unrelated influenza B virus from their lungs. Mice immunized with rNP precipitated with alhydrogel, that subsequently developed significant resistance to virus infection, failed to generate detectable levels of class I major histocompatibility complex (MHC)-restricted killer cells. Furthermore, B10.A(5R) mice that are non-responders for NP-specific class I killer cells could also be protected by immunization with rNP. In contrast, rNP-immunized mice developed strong proliferative T-cell responses to rNP. These data argue for an important role for helper T cells rather than virus-specific class I cytotoxic T cells in protection against influenza virus infection induced by rNP.

Animals↗
PubMed2000

Alu-DNA repeat-binding protein p68 is a part of Alu-RNA containing alpha-RNP.

An Alu-DNA repeat-binding protein with a molecular mass of 68 kDa (p68) is identified in the somatic human cell nucleoplasm. Gel mobility shift assay (GMSA), South-western blotting and affinity purification on DNA attached to the carrier were used in the identification. GMSA revealed multiple complexes with the exponential dependence of their relative mobility. A narrow binding site of the p68 was revealed using synthetic oligonucleotides. It is located between the A-box and B-box of the RNA polymerase III promoter and is identical to that reported for the Alu-binding protein from human spermatozoids. The same narrow binding site, the similarity of the isolation procedure from germ and somatic cells, and similar binding properties and molecular masses suggest homology of the two proteins. Antibodies raised against Alu-protein complexes led to hypershift of the complexes in GMSA and stained p68 in active fractions in human spermatozoids and in Alu-RNA-containing alpha-RNP particles. Immunofluorescence of a HeLa cell monolayer revealed an intranuclear dot pattern with the dots corresponding to euchromatin areas and some dots located at the cell periphery in the cytoplasm. alpha-RNP particles bound Alu-DNA in vitro and contained p68 as shown using the immunogold procedure. Alu-DNA binding activity was revealed in cytoplasm as well as in nucleoplasm. The possible nature of the main Alu-DNA binding protein and its involvement in the particle structure are discussed.

Alu Elements↗
PubMed1991

The complexity of 75S premessenger RNA in balbiani ring granules studied by a new RNA band retardation assay.

Under normal growth conditions, Balbiani ring granules constitute premessenger ribonucleoprotein (RNP) particles synthesized in two chromosomal puffs, Balbiani ring (BR) 1 and 2, in the larval salivary glands of Chironomus tentans. At least three genes encoding 75S RNA are present in these two BRs: one in BR1 and two in BR2 (BR2.1 and BR2.2). The complexity of BR granule 75S RNA was studied by agarose gel electrophoresis under non-denaturing conditions. We recorded three main bands, designated I, II and III. Experiments with denaturing gels demonstrated that the differences in migration reflected mainly, but not exclusively, conformational differences. Northern blotting experiments showed that band I contained BR1 sequences, band II contained BR2.1 sequences, and band III contained BR2.2 sequences. To study whether additional genes contributed to the BR granule 75S RNA, an RNA band shift assay was developed. When an oligodeoxyribonucleotide complementary to repetitive BR1 and BR2.2 sequences was hybridized to 75S RNA prior to electrophoresis, bands I and III were retarded but not band II. An oligonucleotide complementary to a repetitive BR2.1 sequence only shifted band II. Since no detectable 75S RNA remained unchanged in these experiments, and all bands were identified by Northern blotting, all the BR granules are likely to originate from the BR1, BR2.1 and BR2.2 genes; no additional genes have to be invoked. Possible applications of the new RNA band shift assay are discussed.

Animals↗
PubMed2001

A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs.

The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.

Adenosine Triphosphate↗
PubMed1997

Intracellular localization and nucleocytoplasmic transport of Ro RNP components.

The Ro and La autoantigens, which were originally identified by sera from autoimmune patients, consist of RNA-protein complexes (RNPs), in which the protein components carry most of the autoantigenic determinants. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY1, hY3, hY4 and hY5 [35], associated with several proteins, Ro60, Ro52 and La [8, 35, 106] and possibly other, yet unidentified polypeptides. Controversial data have been published on the association of the protein calreticulin with Ro RNPs [53, 58, 75, 78], but recently in vitro evidence has been obtained that non-phosphorylated calreticulin is able to interact with hY RNAs directly [17]. The physiological significance of this interaction remains to be established. In addition to its association with hY RNAs, La is (in most cases transiently) associated with all other RNA polymerase III transcripts. The hY RNAs are the only known cellular RNAs stably bound by La. While the Ro RNPs were found to reside mainly in the cytoplasm, the other La RNPs are mainly nuclear. In this review recent progress made on the intracellular localization and transport of Ro RNP components and of assembled Ro RNPs will be discussed.

Animals↗