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PubMed1995

Structural interaction between the nuclear pore complex and a specific translocating RNP particle.

The transport of Balbiani ring (BR) premessenger RNP particles in the larval salivary gland cells of the dipteran Chironomus tentans can be followed using electron microscopy. A BR RNP particle consists of an RNP ribbon bent into a ringlike structure. Upon translocation through the nuclear pore complex (NPC), the ribbon is straightened and enters the central channel of the NPC with the 5' end of the transcript in the lead. The translocating ribbon is likely to interact with the central channel but, in addition, the remaining portion of the ribbon ring makes contact with the periphery of the NPC. To determine the nature of this latter interaction, we have now studied the connections between the RNP particle and the border of the NPC during different stages of translocation using electron microscope tomography. It was observed that the 3' terminal domain of the ribbon always touches the nuclear ring of the NPC, but the precise area of contact is variable. Sometimes also a region on the opposite side of the ribbon ring reaches the nuclear ring. The pattern of contacts could be correlated to the stage of translocation, and it was concluded that the particle-nuclear ring interactions reflect a rotation of the ribbon ring in front of the central channel, the rotation being secondary to the successive translocation of the ribbon through the channel. The particle's mode of interaction with the NPC suggests that the initial contact between the 5' end domain of the ribbon and the entrance to the central channel is probably crucial to accomplish the ordered translocation of the premessenger RNP particle through the NPC.

Animals↗

PubMed1988

Functional and structural analysis of the ribonucleoprotein complexes of different human influenza virus strains.

Ribonucleoprotein (RNP) cores were prepared from various strains of human influenza virus by treating the purified or spikeless virus particles with non-ionic detergents such as Nonidet P-40 and centrifugation in continuous linear glycerol gradients. In addition to RNA, the purified complexes contained viral nucleoprotein (NP) and the three P proteins (PA, PB1, PB2) as determined by polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. Contaminations with other viral polypeptides, especially HA1, HA2 and M were below 1%. All RNP complexes were transcriptionally active in vitro. Comparison of the polymerase activity of purified complexes revealed considerable differences depending not only on the content of polymerase proteins. The activity of RNP complexes was enhanced for all strains tested by adding of ApG.

DNA-Directed RNA Polymerases↗

PubMed1999

The 5S rRNA is associated with Ro60 ribonucleoprotein and is co-precipitated with hYRNAs by anti-Ro antibodies.

Ro particles are conserved molecules that contain a YRNA and various Ro proteins, which are recognized by autoimmune sera from patients with lupus erythematosus or Sjögren's syndrome. The Ro60 ribonucleoprotein (RNP) forms complexes with certain 5S rRNAs, in such a manner that Ro60 could participate in the control of 5S rRNA production. The present studies were carried out to explore the interaction of Ro components, and to address the question whether Ro60 RNP binds simultaneously 5S rRNA and hYRNA. Anti-Ro60 antibodies were used to immunoprecipitate the RNA. Immunoprecipitates were reverse transcribed with specific oligonucleotides and the resulting cDNAs from 5S and hY4 were amplified by PCR. We found that 5S rRNA is complexed with hY4 and hY5 RNAs by means of the Ro60 RNP. Moreover, by in situ hybridization assays we were able to demonstrate that these molecules have a similar nuclear distribution. According to these results, it seems reasonable to assume that the Ro60 protein could be involved in ribosome assembly.

Antibodies, Monoclonal↗

PubMed2000

Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor.

The mechanism by which a signal recognition particle (SRP) and its receptor mediate protein targeting to the endoplasmic reticulum or to the bacterial plasma membrane is evolutionarily conserved. In Escherichia coli, this reaction is mediated by the Ffh/4.5S RNA ribonucleoprotein complex (Ffh/4.5S RNP; the SRP) and the FtsY protein (the SRP receptor). We have quantified the effects of 4.5S RNA on Ffh-FtsY complex formation by monitoring changes in tryptophan fluorescence. Surprisingly, 4.5S RNA facilitates both assembly and disassembly of the Ffh-FtsY complex to a similar extent. These results provide an example of an RNA molecule facilitating protein-protein interactions in a catalytic fashion.

Bacterial Proteins↗

PubMed1994

Disassembly of influenza C viruses, distinct from that of influenza A and B viruses requires neutral-alkaline pH.

Influenza C (Flu C) viruses comprise an internal ribonucleoprotein (RNP) and an outer lipoprotein envelope with surface spike glycoproteins and the M1 protein matrix. The lipoprotein envelope and spike glycoproteins are solubilized by nonionic detergent in a pH-independent manner. In contrast, disassembly of the M1 protein matrix appears to depend on pH. Treatment of Flu C viruses with nonionic detergent in neutral or alkaline medium (pH 9.0-7.4) results in disintegration of the virion M1 matrix and leads to a significant release of RNP free of the M1 protein. In acidic medium (pH 6.0-5.0), the M1 matrix is not removed and the viral core-like complex of RNP along with the M1 matrix cover is released. Since Flu A and B viruses were characterized by acid-dependent disassembly of the virion M1 matrix, Flu C viruses seem to resemble the paramyxoviruses, which also show a neutral-alkaline pH dependence on matrix disintegration. These observations suggest that uncoating mechanisms of influenza C viruses and paramyxoviruses in target cells may be similar.

Acids↗

PubMed2000

The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans.

In metazoans, most pre-messenger RNAs contain introns that are removed by splicing. The spliced mRNAs are then exported to the cytoplasm. Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known. Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref. 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing. In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes. Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP. Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo. Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus. We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.

Animals↗

PubMedDate not supplied

[Biochemical features of protein matrix M1 of the influenza C virus].

Influenza viruses A, B, and C belonging to Orthomyxoviridae comprise an internal ribonucleoprotein (RNP) and an outer lipoprotein envelope with surface spike glycoproteins and the M1 protein matrix. The lipoprotein envelope and spike glycoproteins are solubilized by nonionic detergent treatment in a pH-independent manner. In contrast, disassembly of the M1 protein matrix appears to depend on pH. Treatment of influenza C viruses with nonionic detergent in neutral or alkaline medium (pH 9.0-7.2) results in disintegration of the virion M1 matrix and leads to a significant release of RNP free of the M1 protein. In acidic medium (pH 6.0-5.0) the M1 matrix fails to be removed and the viral core-like complex of RNP along with the M1 matrix cover is released. Since influenza A and B viruses were characterised by acid-dependent disassembly of the virion M1 matrix, influenza C viruses seem to be more resemble the paramyxoviruses, which also show a neutral-alkaline pH dependence of the matrix disintegration. These observations suggest that uncoating of influenza C viruses and paramyxoviruses in target cells may have similar events.

Hydrogen-Ion Concentration↗

PubMed2003

3D structure of the influenza virus polymerase complex: localization of subunit domains.

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP-mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP.

Image Processing, Computer-Assisted↗

PubMed1983

The major ribonucleoprotein-associated protein kinase of vesicular stomatitis virus is a host cell protein.

Ribonucleoprotein particles (RNPs) of vesicular stomatitis virus (VSV) were fractionated by column chromatography through Fractogel TSK HW-55F and by centrifugation through KCl sucrose. Analyses of fractions for protein content and for protein kinase activity indicated that the major peak of kinase activity did not correspond exactly with any of the VSV-specific proteins. Neither anti-NS nor anti-M IgG preparations inhibited protein kinase activity, and IgG did not act as an exogenous phosphate acceptor. Reconstitution of an RNP-enzyme complex did not result in a restoration of protein kinase activity. In vitro translation of VSV-specific poly(A)-containing RNA did not result in any detectable production of kinase activity. Thus, the major RNP-associated kinase is a host cell protein which is tightly bound to the RNP particle.

Centrifugation, Density Gradient↗

PubMed1996

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

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

Animals↗

PubMed2001

Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains.

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.

Amino Acid Sequence↗

PubMed2001

RNP localization and transport in yeast.

The localization of mRNAs is used by various types of polarized cells to locally translate specific proteins, which restricts their distribution to a particular sub-region of the cytoplasm. This mechanism of protein sorting is involved in major biological processes such as asymmetric cell division, oogenesis, cellular motility, and synapse formation. With the finding of localized mRNAs in the yeast Saccharomyces cerevisiae, it is now possible to benefit from the powerful yeast laboratory tools to explore the molecular basis of RNA localization. Because mRNA transport and localization in yeast share many features with RNA localization in higher eukaryotes, including the formation of a large ribonucleoprotein (RNP) localization complex, the requirement of a polarized cytoskeleton and molecular motors, and the role of nuclear RNA-binding proteins in cytoplasmic localization, the yeast can be used as a paradigm for unraveling the molecular aspects of this process. This review summarizes the current knowledge on RNP transport and localization in yeast.

Actins↗

PubMed2002

Sequence-specific interaction of U1 snRNA with the SMN complex.

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem--loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.

Animals↗

PubMed2004

An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay.

The specification of both the germ line and abdomen in Drosophila depends on the localization of oskar messenger RNA to the posterior of the oocyte. This localization requires several trans-acting factors, including Barentsz and the Mago-Y14 heterodimer, which assemble with oskar mRNA into ribonucleoprotein particles (RNPs) and localize with it at the posterior pole. Although Barentsz localization in the germ line depends on Mago-Y14, no direct interaction between these proteins has been detected. Here, we demonstrate that the translation initiation factor eIF4AIII interacts with Barentsz and is a component of the oskar messenger RNP localization complex. Moreover, eIF4AIII interacts with Mago-Y14 and thus provides a molecular link between Barentsz and the heterodimer. The mammalian Mago (also known as Magoh)-Y14 heterodimer is a component of the exon junction complex. The exon junction complex is deposited on spliced mRNAs and functions in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs with premature translation-termination codons. We show that both Barentsz and eIF4AIII are essential for NMD in human cells. Thus, we have identified eIF4AIII and Barentsz as components of a conserved protein complex that is essential for mRNA localization in flies and NMD in mammals.

Active Transport, Cell Nucleus↗

PubMed1997

Functionally interacting telomerase RNAs in the yeast telomerase complex.

The ribonucleoprotein (RNP) enzyme telomerase from Saccharomyces cerevisiae adds telomeric DNA to chromosomal ends in short increments both in vivo and in vitro. Whether or not telomerase functions as a multimer has not been addressed previously. Here we show, first, that following polymerization, the telomerase RNP remains stably bound to its telomeric oligonucleotide reaction product. We then exploit this finding and a previously reported mutant telomerase RNA to demonstrate that, unexpectedly, the S. cerevisiae telomerase complex contains at least two functionally interacting RNA molecules that both act as templates for DNA polymerization. Here, functional telomerase contains at least two active sites.

Base Sequence↗

PubMed1985

Regulation of viral transcription by the matrix protein of vesicular stomatitis virus probed by monoclonal antibodies and temperature-sensitive mutants.

The ability of the matrix (M) protein of wild-type vesicular stomatitis virus (VSV) to regulate viral transcription was studied with monoclonal antibodies and temperature-sensitive (ts) mutants in complementation group III, the M proteins of which are restricted in transcription inhibition. The marked inhibition of transcription by VSV ribonucleoprotein (RNP) cores complexed with M protein (RNP/M) was reversed by antibody to epitope 1. Antibodies to epitopes 2 and 3 not only failed to reverse the transcription-inhibitory activity of isolated M protein but actually increased M-protein inhibition of transcription in a reconstituted system. Monoclonal antibodies to epitopes 2 and 3 strongly bound to M proteins from all wild-type and ts-mutant virions, but monoclonal antibody to epitope 1 completely failed to bind to the M protein of ts023(III) even though it reacted strongly with M proteins of mutants tsG31(III) and tsG33(III). The M protein of a tsO23 revertant (R11) completely recovered its capacity to inhibit transcription and to bind monoclonal antibody to epitope 1, whereas the M proteins of three other revertants remained restricted in their capacity to inhibit transcription and to bind monoclonal antibody to epitope 1. These studies indicate that exposure of epitope 1 on the surface of M protein is essential for inhibiting transcription by VSV RNP cores.

Antibodies, Monoclonal↗

PubMed1995

Components of the nucleolar processing complex (Pre-rRNA, fibrillarin, and nucleolin) colocalize during mitosis and are incorporated to daughter cell nucleoli.

We have traced in onion root cells the mitotic course of rRNA, of the RNA synthesized in the G2 period of the preceding interphase, and of the nucleolar proteins fibrillarin and nucleolin. The rRNA was detected by ultrastructural in situ hybridization with a rDNA probe capable of hybridizing mature rRNAs and also the intermediate forms of pre-rRNA processing. The RNA synthesized in the preceding G2 (which, according to classical data, is mostly rRNA) was revealed by autoradiography on synchronous cells labeled in G2 by tritiated uridine. Fibrillarin was detected by immunofluorescence in both mammalian and onion cells; the results in the latter cells were compared with those obtained after AgNOR staining. Electron microscopical immunocytochemistry was used to detect fibrillarin and nucleolin in onion cells. In all cases, following nucleolar dispersion in prophase, the signal was detected in the chromosome periphery during metaphase and anaphase, in irregular fibrillar masses located between chromosomes in ana-telophase, in prenucleolar bodies during telophase, and in the newly formed nucleoli, after nucleologenesis. Moreover, as expected, ribosomes appeared labeled after in situ hybridization, but a dispersed cytoplasmic labeling was observed in all experiments, mainly during metaphase and anaphase. These results demonstrate that nucleolar components involved in pre-rRNA processing, including rRNA itself, probably in an incompletely processed form, are transferred from the parental to the daughter cell nucleoli by means of transient structures, such as the perichromosomal sheath and prenucleolar bodies. Since these macromolecular components are assembled in the interphase nucleolus, forming the RNP processing complex, their colocalization during mitosis in the same transient structures strongly suggests that at least a subset of these complexes does not disaggregate during cell division, but remains assembled and becomes incorporated to the new nucleolus. Therefore, ribosome biogenesis restarts not only after mitosis at the level of transcription, but also at the intermediate levels of pre-rRNA processing.

Allium↗

PubMed1987

Effect of enzymatic treatments on RNP and Sm antigenic reactivities--I. Loss of RNP but increase of Sm antigenic reactivity after RNase treatment of nuclear extract.

Changes of RNP and Sm antigenic reactivities of a nuclear extract after enzymatic treatments were studied and quantified by the ELISA test. After RNase treatment of the nuclear extract, about a 300% increase of the Sm antigenic reactivity and more than a 95% decrease of RNP antigenic reactivity was found. Data from RNP-depleted nuclear extracts and column fractionation show that the increase in Sm antigenic reactivity after RNase treatment mainly comes from the RNP-Sm complex.

Antibodies, Antinuclear↗