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PubMed2006

A fast and efficient procedure to produce scFvs specific for large macromolecular complexes.

We have expanded the application of antibody phage display to a new type of antigen: ribonucleoprotein (RNP) complexes. We describe a simple and efficient method for screening antibodies specific for large intact RNPs and individual components. We also describe a fast and easy method to overcome the abundance of amber stop codons in the positive phage clones. The resulting antibodies have been used in ELISA and Western blot analysis.

Antibodies, Monoclonal↗
PubMed2002

Modeling of a possible evolutional process from a ribozyme to a catalytic RNP.

A model process for molecular evolution from an RNA enzyme to a catalytic RNA-protein complex (RNP) is proposed. In the model, one RNA-RNA interaction in the enzyme is replaced by an RNA-protein interaction via an intermediary state where the original RNA-RNA and newly introduced RNA-protein interaction co-exist. To test the model, a catalytic RNP was designed and examined by employing the Tetrahymena ribozyme.

Animals↗
PubMed1989

The sphere organelle contains small nuclear ribonucleoproteins.

We show by immunofluorescence microscopy of amphibian oocyte nuclei that small nuclear ribonucleoproteins (snRNPs) occur in lampbrush chromosome loops, in a few dozen extrachromosomal organelles previously described as "spheres," and in thousands of smaller granules. Spheres are variable in size (up to approximately 20 microns in diameter in the newt Notophthalmus and approximately 10 microns in the frog Xenopus) and are easily distinguishable from nucleoli by morphology and composition. Spheres occur both free in the nucleoplasm and attached to specific chromosome loci, the sphere organizers. Oocyte nuclei of a cricket and a spider contain essentially similar organelles, suggesting that spheres may be common throughout the animal kingdom. We suggest that spheres play a role in the assembly of snRNP complexes for the nucleus comparable to the way that nucleoli assemble ribosomal RNP complexes for the cytoplasm.

Animals↗
PubMed2006

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

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 associated with the translation initiation eIF4F complex that may affect complex formation were identified and quantitated under distinct growth conditions. Site-specific phosphorylation of eIF4E and eIF4G and elevated levels of eIF4G:eIF4E complexes in phorbol ester treated HEK293 cells, and in serum-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 complexes, for functions related to RNP assembly, remodeling and/or transition from active translation to mRNA degradation. Our results demonstrate that our quantitative proteomic strategy can be applied to the identification and quantitation of protein complex components in human cells grown under different conditions.

Amino Acid Sequence↗
PubMed1983

Fine structure analysis of Pichinde virus nucleocapsids.

The structure and organization of the ribonucleoprotein (RNP) complex of an arenavirus, Pichinde virus, was investigated. The basic configuration of the RNP was found to be a linear array of globular subunits or nucleosomes, 4 to 5 nm in diameter, that represent individual molecules of the major N polypeptide. This filament appears to fold progressively through a number of intermediate helical structures, 12 to 15 nm in diameter, that reveal an increasing number of nucleosomes associated with each turn of the helix. They range from a fragile configuration of two or three nucleosomes per turn to a more stable fibre in which the nucleosomes cannot be resolved. The strands were shown to form closed circles and it appeared that the twisting of these circular forms resulted in the formation of 20 nm-thick fibres which were seen in isolated viral core structures. The association of these RNP structures with other viral components is discussed.

Arenaviridae↗
PubMed1996

[The association of specific RNPs with the EGF receptor in A-431 cells].

One of alpha-RNP proteins (38 kDa alpha-protein) has been revealed as a target of the EGF receptor-associated protein kinase. A specific association of alpha-RNP complexes with the EGF receptor has been demonstrated. The involvement of alpha-RNP in the transduction of the EGF-induced signal has been suggested.

Carcinoma, Squamous Cell↗
PubMed1994

Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes.

Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.

Animals↗
PubMed2005

TbMP42, a protein component of the RNA editing complex in African trypanosomes, has endo-exoribonuclease activity.

RNA editing in trypanosomatids is catalyzed by a high molecular mass RNP complex, which is only partially characterized. TbMP42 is a 42 kDa protein of unknown function that copurifies with the editing complex. The polypeptide is characterized by two Zn fingers and a potential barrel structure/OB-fold at its C terminus. Using recombinant TbMP42, we show that the protein can bind to dsRNA and dsDNA but fails to recognize DNA/RNA hybrids. rTbMP42 degrades ssRNA by a 3' to 5' exoribonuclease activity. In addition, rTbMP42 has endoribonuclease activity, which preferentially hydrolyzes non-base-paired uridylate-containing sequences. Gene silencing of TbMP42 inhibits cell growth and is ultimately lethal to the parasite. Mitochondrial extracts from TbMP42-minus trypanosomes have only residual RNA editing activity and strongly reduced endo-exoribonuclease activity. However, all three activities can be restored by the addition of rTbMP42. Together, the data suggest that TbMP42 contributes both endo- and exoribonuclease activity to the editing reaction cycle.

Animals↗
PubMed1985

Transcription boundaries of U1 small nuclear RNA.

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

Base Sequence↗
PubMed1983

In vitro assembly of a pre-messenger ribonucleoprotein.

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

Adenoviruses, Human↗
PubMed1982

Ultrastructure of free ribonucleoprotein complexes in spread mammalian nuclei.

Mouse erythroleukemia cell nuclei obtained by three different methods were spread for electron microscopy under low ionic conditions. It was found that this procedure allows the observation of free large ribonucleoprotein (RNP) complexes released from the nuclei during the centrifugation. The morphology of these complexes was readily affected by the conditions of cell treatment and spreading. Two extreme forms of free nuclear RNP structures were obtained, both consisting of spherical particles with diameters of approximately 17-20 nm. The first type was of loosened complexes of irregularly assembled particles interconnected with RNA fibrils. The second represented tightly packed particles forming mostly branched structures. The latter structures appeared to be closer to the native form of the nuclear RNP particles, differing from polyribosomes by their characteristic branching and stability in EDTA solutions.

Animals↗
PubMed1990

Quantitative determination that one of two potential RNA-binding domains of the A protein component of the U1 small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of U1 RNA.

Many RNA-associated proteins contain a ribonucleoprotein (RNP) consensus octamer encompassed by a conserved 80 amino acid sequence, which we have termed an RNA recognition motif (RRM). RRM family members contain either one (class I) or multiple (class II) copies of this motif. We report here that a class II component of the U1 small nuclear RNP (snRNP), the A protein of U1 snRNP (U1snRNP-A), contains two RRMs (RRM1 and -2), yet has only one binding domain (RRM1) that interacts specifically with stem-loop II of U1 RNA. Quantitative analysis of binding affinities of fragments of U1snRNP-A demonstrated that an 86-amino acid polypeptide was competent to bind to U1 RNA with an affinity comparable to that of the full-length protein (Kd approximately 80 nM). The carboxyl-terminal RRM2 of U1snRNP-A did not bind to U1 RNA and may recognize an unidentified heterologous RNA. We propose that class II proteins may function as bridges between RNA components of RNP complexes such as the spliceosome.

Binding Sites↗
PubMed2001

Telomerase and cancer.

Telomerase, a eukaryotic ribonucleoprotein (RNP) complex, contains both an essential RNA and a protein reverse transcriptase subunit. By reverse transcription, the telomerase RNP maintains telomere length stability in almost all cancer cells. Over the past few years there has been significant progress in identifying the components of the telomerase holoenzyme complex and the proteins that associate with telomeres, in order to elucidate mechanisms of telomere length regulation. This review covers recent advances in the field including the use of telomerase in cancer diagnostics and an overview of anti-telomerase cancer therapeutic approaches.

Animals↗
PubMed1980

[Presence of antigenic determinants in mRNA-containing RNP particles of immune B-lymphocytes].

An iodinated erythrocyte antigen isolated from sheep erythrocytes (125I-EAG) was injected to the BALB/c or CBA mice in a single dose of 20-25 mkg as for protein. The antigen determinants were detected by incorporation of a radioactive label into spleen B-lymphocytes on the 1sh-5th post-immunization days. It was shown that the T-cells contain no label and that 2/3 of the radioactivity of B-lymphocyte suspension of the 125I-EAG-immunized mice are detected in the cell nuclei within mRNP. During electrophoresis of the particles in 2,5% polyacrylamide gel nuclear mRNP isolated from B-lymphocytes of mice immunized with cold EAG are adsorbed by an immunoadsorbent containing antibodies against sheep erythrocytes. Fractionation of 125I-cytoplasmic extract obtained from 125I-EAG-immunized mouse B-lymphocytes in sucrose gradient revealed that the radioactive label was detected in the same two regions of the sucrose gradient, which is occupied by light RNP complexes containing mRNA and polyribosomes. Under experimental conditions allowing to detect the transitions of mRNP complexes into polyribosomes, a shift of the 125I-labelled material (probably together with the RNP particles) towards polyribosomes was observed. Thus, the antigen determinants are detected as part of nuclear and cytoplasmic mRNP of B-lymphocytes during the whole cycle of the primary immune response and are detected within the composition of cytoplasmic mRNP during the immunoglobulin polypeptide chain synthesis.

Animals↗
PubMed1983

Clinical characteristics of patients with rheumatic disorders who possess antibodies against ribonucleoprotein particles.

IgG was purified from 91 serum samples obtained from patients with systemic rheumatic diseases and examined for the presence of antinuclear antibodies reactive with small ribonucleoprotein (RNP) complexes. Antibody specificity was determined by autoradiographic detection of 32P labeled RNA which had been separated by polyacrylamide gel electrophoresis. This RNA was extracted from immune complexes formed by adding purified IgG to a homogenate of 32P labeled HeLa cells. Anti-RNP antibodies were detected in 70% of these samples. Specific antibodies detected were anti-(U1)-RNP (35%), anti-Sm (30%), anti-Ro (24%), and anti-La (10%). Other antibodies (9%) were reactive with structures containing 5S and 5.8S RNA or 4S RNA. There was a significant relationship between the presence of anti-RNP antibodies in patients with systemic lupus erythematosus (SLE) (78%) as opposed to their presence in patients with non-SLE rheumatic disorders (45%). However, taken individually, anti-Ro was the only antibody whose presence in SLE patients (30%) was significantly greater than its presence in non-SLE patients (5%). Vasculitis was the only clinical manifestation found to correlate with the presence of a particular antibody, anti(U1)RNP. There is some suggestion of a racial distribution of antibody types.

Antibody Specificity↗
PubMed1983

In vitro inhibition of negative strand virus transcriptase activity by proteins soluble in acidic chloroform-methanol.

The effect of proteins soluble in acidic chloroform-methanol (ACMS proteins) on the transcriptase activity of virus ribonucleoproteins (RNPs) in vitro has been studied. Experiments with ACMS membrane (M) proteins from type A and B orthomyxoviruses, as well as from vesicular stomatitis virus, showed that inhibition of the viral RNP transcriptase activity occurred when they interacted with M proteins isolated from viruses of a different serotype, or even of a different family. The presence of ACMS proteins capable of inhibiting the transcriptase activity of orthomyxovirus RNP in vitro was also detected in human blood plasma and among proteins produced by human leukocytes. Determination of the minimum concentration of M protein inhibiting the RNP transcriptase activity, and analysis of the fowl plague virus M protein-RNP complex formed in the in vitro system, showed that the M protein was capable of inhibiting RNP transcriptase activity at a M:RNP ratio of 0.1 to 0.2:1.

Blood Proteins↗
PubMed2000

Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo.

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Animals↗