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Results for “RNP complex”

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PubMed1988

Estradiol-stimulated nuclear ribonucleoprotein transport in the rat uterus: a molecular basis.

The present investigation probes the intranuclear molecular changes that serve to link the nuclear binding of estradiol with the hormone-stimulated ribonucleoprotein (RNP) transport in the rat uterus. Within 2 min of in vitro exposure of isolated uterine nuclei to 10 nM 17 beta-estradiol a Mg2+-dependent nuclear ATPase becomes activated and reaches its peak activity. This is immediately followed by a phase of ATP resynthesis. This newly synthesized ATP serves as the substrate for the nuclear protein kinases. Cyclic AMP inhibits this ATP resynthesis and, as a consequence, prevents the estradiol-stimulated nuclear protein kinase activity and the exit of the RNP-estradiol complex from the nuclei. cGMP is stimulatory to the estradiol-mediated nuclear ribonucleoprotein transport.

Adenosine Triphosphate↗

PubMed2002

Assisted RNP assembly: SMN and PRMT5 complexes cooperate in the formation of spliceosomal UsnRNPs.

Although spliceosomal Sm proteins can assemble spontaneously onto UsnRNA in vitro, this process requires assisting factors in vivo. SMN, the protein involved in spinal muscular atrophy, is part of a complex that contains the Sm proteins and serves as a critical factor for this reaction. Here, we have reconstituted the SMN-dependent assembly of UsnRNPs in vitro. We demonstrate that the SMN complex is necessary and sufficient for the assembly reaction. The PRMT5 complex, previously implicated in methylation and storage of Sm proteins, interacts with the SMN complex and enhances its activity in an ATP-dependent manner. These data uncover the SMN-PRMT5 complex as a functional entity that promotes the assisted assembly of spliceosomal UsnRNPs, and potentially other, RNA-protein complexes.

Animals↗

PubMed1982

Kinetics of synthesis of influenza virus ribonucleoprotein structures.

The synthesis of influenza virus ribonucleoprotein structures (RNPs) in infected chick embryo cells was analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium deoxycholate which resolves the RNPs into five size classes. A relatively small proportion of total RNPs accumulated in the nucleus but free NP protein was found there in large amounts over the period 1.5 to 4 h post-infection. In contrast, by 4 h post-infection, all cytoplasmic NP was complexed into RNP structures. At early times, during at 15 min pulse of (35S]methionine, nearly all the newly synthesized NP was incorporated into RNPs but by 4 h the majority of pulse-labelled NP was present as free protein. However, the proportion of free NP: NP in RNPs remained constant over the 1.5 to 4 h post-infection period, indicating that there was a delay before the NP synthesized later in infection was assembled into RNP structures. Individual RNP size classes were predominantly cytoplasmic and accumulated at similar rates but were not produced in equimolar amounts. The rates of synthesis of individual RNPs were in general agreement with their rates of accumulation with the remarkable exception of RNP d (containing RNA 7, the matrix protein gene). This was synthesized nearly 10-fold faster but accumulated at the same rate as the other RNPs. Possibly RNP d is more rapidly degraded than the other RNPs.

Animals↗

PubMed1992

Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure.

A temperature-sensitive lethal mutation in Saccharomyces cerevisiae, prp20-1, causes defects in several different steps in mRNA metabolism, including mRNA 3'-end formation, transcription initiation, and mRNA transport. Previous work has demonstrated that prp20 mutants are defective in actin pre-mRNA splicing. PRP20 is related, both in structure and function, to the RCC1 gene of mammals and the PIM1 gene of Schizosaccharomyces pombe, both of which appear to regulate entry into mitosis and chromosome condensation. In this report we demonstrate that, after a shift of prp20 mutants to the restrictive temperature, transcripts of several genes (CUP1, CYH2, and GAL10) are produced that extend 1-10 kb beyond their normal polyadenylation sites. The failure in 3'-end formation occurs within 1-2 min of the temperature shift. Transcription initiation also is disrupted, in that initiation sites upstream of the normal cap site are used. mRNA transport from nucleus to cytoplasm also is perturbed: In situ hybridization using an oligo(dT) probe demonstrates accumulation of poly(A) in the nucleus, consistent with the accumulation of longer bulk poly(A) (up to approximately 90-100 nucleotides) and with a failure to transport newly synthesized RNA to the cytoplasm. We demonstrate that prp20 and rna1 mutants are very similar, if not identical, with respect to each of these biochemical phenotypes. In light of the putative role of PRP20 in mitotic control, our results suggest a common step in that process and multiple steps in mRNA synthesis and maturation. We speculate that the perturbations in mRNA processing are the result of effects on the chromatin-nascent RNP-transcription complex or misregulation of a cell cycle component that modifies multiple mRNA-processing activities.

Base Sequence↗

PubMed1976

On the mode of activation of sequestered messengers in Artemia salina.

Activation of the dormant embryos of Artemia salina was marked by a rapid increase in 32P uptake which reached a stationary phase after 6 h of activation. The increase in 32P uptake by whole cysts was paralleled by its incorporation into nucleotides. Fractionation of acid-soluble nucleotides and alkaline hydrolysate of nucleic acids on Dowex-1-formate column revealed the 32P radioactivity to be exclusively localised in AMP. Analysis of the labelled RNA species extracted at different stages of development indicated a preferential labelling of small molecular weight species till the emergence of the embryos, followed by the de novo synthesis of messenger and stable RNA species in later stages of development. During early development, polyadenylated RNA species were localised in the particulate fraction sedimenting at 16,000 rpm and their location shifted to the soluble fraction as development proceeded. Activation of performed messengers by phosphorylation of the adenylate residue of their poly A stretches and translocation of the capacitated messengers to the cytosol via a RNP-membrane complex is proposed as a trigger of embryonic differentiation.

Animals↗

PubMed1999

Assessing the function of the Ro ribonucleoprotein complex using Caenorhabditis elegans as a biological tool.

The Ro ribonucleoprotein complex (Ro RNP) was initially described as an autoimmune target in human diseases such as systemic lupus erythematosus and Sjögren's syndrome. In Xenopus and human cells, its general structure is composed of one major protein of 60 kDa, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Although no function has been assigned to the Ro RNP, Ro60 has been shown to bind mutant 5S ribosomal RNA (rRNA) molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. Ro60 has also been shown to participate in the regulation of the translational fate of the L4 ribosomal protein mRNA by interacting with the 5' untranslated region, again suggesting its possible implication in ribosome biogenesis. To identify the function of Ro RNP, we have taken a genetic approach in the nematode Caenorhabditis elegans. As such, we characterized the gene encoding the protein ROP-1, the homologue of the human Ro60 protein. Here, we review the phenotypic analysis of C. elegans rop-l(-) mutants and integrate these results into a model for the function of the Ro RNP particle.

Animals↗

PubMed1991

Molecular characterization of the Ro/SS-A autoimmune response.

Human Ro/SS-A (Ro) antibodies (anti-Ro) are frequently seen in the circulation of patients with several subsets of photosensitive cutaneous lupus erythematosus (LE) and related disorders. Experimental observations have suggested that anti-Ro reacting with Ro antigens that are expressed abberantly within the epidermis might play a causal role in LE-specific skin disorders such as subacute cutaneous LE (SCLE) and neonatal LE. In this article, we will review the progress that has recently been achieved toward gaining a better understanding of the molecular configuration, genetic regulation, and function of the Ro ribonucleoprotein (RNP) antigenic complex as well as the autoimmune response with which it is associated. Work in this area has recently revealed the existence of multiple Ro autoantigen-bearing polypeptides including a 46-kd Ro protein that is virtually identical to calreticulin, a highly conserved, calcium-binding protein that is normally associated with the endoplasmic reticulum. The implications of these observations with respect to the pathogenesis of anti-Ro-associated LE skin lesions are explored.

Antibodies, Antinuclear↗

PubMed1990

A Trypanosoma brucei small RNP particle containing the 5S rRNA.

In mammalian cells, approximately 50% of the 5S rRNA is found in ribosomes, and the remainder in a small particle, the 5S rRNA/ribosomal protein L5 complex, which is thought to be a precursor in ribosome assembly. Trypanosoma brucei, an African trypanosome, is one of the most primitive eukaryotic organisms which have been studied, and it likewise possesses a 5S rRNA species, a small proportion of which is found in an apparent ribonucleoprotein-(RNP) complex. Like the mammalian RNP particle, the T. brucei particle has a sedimentation coefficient of about 7S in sucrose gradients; unlike its mammalian counterpart, the complex is not disrupted by high salt and can be fractionated in cesium sulfate density gradients at a density characteristic of RNP complexes (1.45 g ml-1). Our studies demonstrate the the T. brucei 7S RNP contains 5S rRNA in association with a 36-kDa rRNA binding protein which not only shares molecular size, but also immunological determinants, with the yeast ribosomal protein YL3, and its mammalian homologue, L5. These results indicate that the RNP complex formed between the 5S rRNA and the 36-kDa ribosomal protein is conserved throughout great evolutionary distances between eukaryotic species.

Animals↗

PubMed1996

Neutralization of HIV type 1 infectivity by serum antibodies from a subset of autoimmune patients with mixed connective tissue disease.

Mixed connective tissue disease (MCTD) is a rheumatic disorder with clinical similarities to HIV-1 infection, and with characteristic autoimmune anti-RNP antibodies specific for the U1 snRNP splicing complex. Anti-RNP antibodies cross-react with the HIV-1 surface, owing to multiple homologies between the gp120/41 envelope complex and the 70K protein of U1 snRNP. A key epitope of 70K, its RNA-binding site, is homologous to a dominant B and T cell epitope in the third variable loop (V3) of gp120. In this study, we tested the ability of anti-RNP sera to inhibit HIV-1 infectivity in vitro. Of nine sera tested, five were 70-99% effective in neutralizing one or more HIV-1 strains. One serum was > 99% effective in neutralizing HIV-1MN, and 86 and 77% effective against the primary isolates HIV-1(CO) and HIV-1(JR-FL), respectively, an efficacy equal to that of a pool of broadly neutralizing antibodies from HIV-1-infected subjects (HIVIG). The mean neutralizing titer of anti-RNP sera against HIV-1(JR-FL) was 3.9-fold higher than that of HIVIG. Neutralizing potency was associated with high reactivity to gp120 by ELISA, and with the presence of serum rheumatoid factor, known to enhance antibody neutralization of other viruses. The current findings provide further evidence that individuals unexposed to HIV-1 may develop immunologic resistance by alternative mechanisms, possibly including molecular mimicry, or exposure to as yet unidentified retroviruses. Thus MCTD, which involves both B and T cell reactivity to self-epitopes homologous to HIV-1, may elucidate new strategies for generating protective immunity to this virus.

Antibodies, Antinuclear↗

PubMed1983

Purification and identification of antigenic polypeptides of Sm and RNP antigens of goat liver.

A ribonucleoprotein complex containing Sm and RNP antigenic activity was isolated from goat liver. The sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the complex to contain four major polypeptides of 80,000, 70,000, 30,000 and 14,000 molecular weights. When the gels were probed for antigenic polypeptides by an enzyme-linked immunosorbent assay and fluorescent antibody method, anti-Sm sera were found to react with polypeptides of 14,000 and 30,000 molecular weights whereas anti-RNP sera recognized 70,000 and 80,000 molecular weight polypeptides with minor reactivity in the polypeptide of 14,000 molecular weight. Sera containing antibodies to both Sm and RNP reacted with all the four polypeptides.

Animals↗

PubMed1991

RNA-protein interactions of stored 5S RNA with TFIIIA and ribosomal protein L5 during Xenopus oogenesis.

We studied the pathway of 5S RNA during oogenesis in Xenopus laevis from its storage in the cytoplasm to accumulation in the nucleus, the sequence requirements for the 5S RNA to follow that pathway, and the 5S RNA-protein interactions that occur during the mobilization of stored 5S RNA for assembly into ribosomes. In situ hybridization to sections of oocytes indicates that 5S RNA first becomes associated with the amplified nucleoli during vitellogenesis when the nucleoli are activity synthesizing ribosomal RNA and assembling ribosomes. When labeled 5S RNA is microinjected into the cytoplasm of stage V oocytes, it migrates into the nucleus, whether microinjected naked or complexed with the protein TFIIIA as a 7S RNP storage particle. During vitellogenesis, a nonribosome bound pool of 5S RNA complexed with ribosomal protein L5 (5S RNPs) is formed, which is present throughout the remainder of oogenesis. Immunoprecipitation assays on homogenates of microinjected oocytes showed that labeled 5S RNA can become complexed either with L5 or with TFIIIA. Nucleotides 11 through 108 of the 5S RNA molecule provide the necessary sequence and conformational information required for the formation of immunologically detectable complexes with TFIIIA or L5 and for nuclear accumulation. Furthermore, labeled 5S RNA from microinjected 7S RNPs can subsequently become associated with L5. Such labeled 5S RNA is found in both 5S RNPs and 7S RNPs in the cytoplasm, but only in 5S RNPs in the nucleus of microinjected oocytes. These data suggest that during oogenesis a major pathway for incorporation of 5S RNA into nascent ribosomes involves the migration of 5S RNA from the nucleus to the cytoplasm for storage in an RNP complex with TFIIIA, exchange of that protein association for binding with ribosomal protein L5, and a return to the nucleus for incorporation into ribosomes as they are being assembled in the amplified nucleoli.

Animals↗

PubMed1986

The relationship between Sm and RNP antigens.

The relationship between the antigenic proteins of Sm and RNP is not clear. To further clarify their relationship, we examined sera found monospecific by counterimmunoelectrophoresis (CIEP) for anti-Sm or anti-RNP with the more sensitive techniques of immunoblotting, radioimmunoprecipitation, or enzyme immunoassay (EIA). The same eight unique problems were precipitated by both anti-Sm and anti-RNP in radioimmunoprecipitation. They had molecular weights (MWs) of 11, 13, 17, 18, 24, 26, 28, and 68 kDa. The 17, 18, and 28 kDa bands were more intense with anti-RNP. Immunoblotting with anti-Sm and anti-RNP also recognized similar proteins with MWs of 14, 17, 25, 28, 29, 30, 36, 38, and 68 kDa. Anti-Sm resulted in more intense 14, 28, 29, and 30 kDa bands, while anti-RNP gave maximum intensity of the 14, 36, 38, and 68 kDa bands. The band intensity pattern differences were more easily appreciated with immunoblotting than with radioimmunoprecipitation. RNase, heat, and urea caused a similar diminution of antigen reactivity with both anti-Sm and anti-RNP on immunoblotting, but eliminated immunoprecipitability only of RNP on immunodiffusion. The great similarities between Sm and RNP suggest several possibilities: Anti-Sm and anti-RNP antibodies coexist in the same patients; and the more sensitive techniques of immunoblotting and radioimmunoprecipitation detect both precipitating and nonprecipitating antibodies while only precipitating antibodies are detected by immunodiffusion. Sm and RNP may represent different determinants on the same macromolecular complex. Sm and RNP may be cross-reacting determinants on distinct molecules.

Adolescent↗

PubMed1985

Interaction of M protein and RNP of fowl plague virus in vitro.

The ability of the fowl plague virus (FPV) M protein to form a complex with FPV RNP and to inhibit the RNP transcriptase activity in vitro depended on NaCl concentration and did not depend on the concentration of nonionic detergents. The results obtained indicate that the M protein-RNP links formed were of an electrostatic rather than a hydrophobic nature. As demonstrated using individual RNP components, vRNA and RNA-free protein structures, M protein formed complexes only with vRNA, and the complex formation was salt-dependent. Analysis of products formed in the in vitro system containing RNP of FPV in the presence of the M protein showed impairment in the transcription of all RNA segments. The degree of inhibition correlated with the size of a segment, transcription of high molecular weight RNA segments being inhibited significantly more than that of low molecular weight RNA segments.

Animals↗

PubMed1983

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.

Newly transcribed heterogeneous nuclear RNA (hnRNA) in the eucaryote cell nucleus is bound by proteins, giving rise to large ribonucleoprotein (RNP) fibrils with an inherent substructure consisting largely of relatively homogeneous approximately 20-nm 30S particles, which contain core polypeptides of 34,000-38,000 mol wt. To determine whether this group of proteins was sufficient for the assembly of the native beaded nucleoprotein structure, we dissociated 30S hnRNP purified from mouse ascites cells into their component proteins and RNA by treatment with the ionic detergent sodium deoxycholate and then reconstituted this complex by addition of Triton X-100 to sequester the deoxycholate. Dissociation and reassembly were assayed by sucrose gradient centrifugation, monitoring UV absorbance, protein composition, and radiolabeled nucleic acid, and by electron microscopy. Endogenous RNA was digested and reassembly of RNP complexes carried out with equivalent amounts of exogenous RNA or single-stranded DNA. These complexes are composed exclusively of groups of n 30S subunits, as determined by sucrose gradient and electron microscope analysis, where n is the length of the added nucleic acid divided by the length of nucleic acid bound by one native 30S complex (about 1,000 nucleotides). When the nucleic acid: protein stoichiometry in the reconstitution mixture was varied, only complexes composed of 30S subunits were formed; excess protein or nucleic acid remained unbound. These results strongly suggest that core proteins determine the basic structural properties of 30S subunits and hence of hnRNP. In vitro construction of RNP complexes using model nucleic acid molecules should prove useful to the further study of the processing of mRNA.

Animals↗

PubMed1982

Protein antigens of the RNA-protein complexes detected by anti-SM and anti-RNP antibodies found in serum of patients with systemic lupus erythematosus and related disorders.

Evidence has been obtained previously indicating that the antigens reacting with the anti-Sm and anti-RNP sera are present as a large complex, and similar protein bands are obtained with both types of sera. Inthe present study, it proved possible to break up this complex using SDS treatment before immunoprecipitation. After such treatment, different protein bands were immunoprecipitated by the two antisera; Sm determinants resided, at least partially, in a 19-kd protein. Sequential immunoprecipitation with and without prior SDS treatment provided further evidence for these specificities and suggested that two classes of particles exist in different tissues, one containing proteins immunoreactive with the Sn and RNP antisera and the other containing proteins immunoreactive only with the Sm antisera. The latter particle contained all the bands seen with the first type except for the absence of the 19-kd band. Nitrocellulose blot analyses confirmed the assignment of the 25- and 16-kd polypeptides to Sm antigenic determinants; analyses for RNP proved les informative by this technique. Some differences in the banding patterns were obtained using cells from different species: the 25-kd Sm band was usually double in human cells and single in rat and rabbit tissue. Methods of extraction also caused some differences which was especially true for the rabbit thymus extract widely used for Sm and RNP studies. Additional immunoreactive bands at 68 and 70 kd also were detected when the Sm and RNP antisera were used in nitrocellulose blot analyses. Furthermore, evidence was obtained for a number of other antibodies in lupus sera which have not as yet been detected by serological methods.

Antibody Specificity↗

PubMed1997

Automated electron tomography of large nuclear RNP (InRNP) particles--the naturally assembled complexes of precursor messenger RNA and splicing factors.

Splicing of nuclear pre-mRNA is an important step in the regulation of gene expression as only correctly spliced mRNAs will be exported to the cytoplasm to function in protein synthesis. Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA, are packaged in multicomponent large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200S region in sucrose gradients, contain all U snRNPs required for pre-mRNA splicing and several protein splicing factors, including U2AF and the SR proteins and can thus be viewed as naturally assembled complexes of pre-mRNA and splicing factors. We have previously reconstructed the three-dimensional image of negatively stained individual lnRNP particles by automated electron tomography. The reconstruction revealed a compact structure, 50 nm in diameter, composed of four major subunits. Here we further analyzed the reconstructed models and the apparent connectivity between the subunits using a new rendering technique. The uniformity of the lnRNP particles was substantiated by measurement of the volume engulfed by their surface. This study further supports the model proposed for the packaging of nuclear pre-mRNAs in lnRNP particles, where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multiintronic pre-mRNAs, and with the fact that the splicing of multiintronic pre-mRNAs does not occur in a sequential manner.

Animals↗

PubMed1983

RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8.

Ribonucleoprotein (RNP) cores with RNA-synthesizing activity were prepared in two fractions, M protein-free and M protein-associated, from detergent-treated influenza virus PR8 by centrifugation through a discontinuous triple gradient of cesium sulfate, glycerol, and NP-40. The M-free RNP was fractionated by phosphocellulose column chromatography into two major RNP forms, A and B, which differed in the content of P proteins, while the M-associated RNP gave only the low P-content Form-B RNP. Starting from the high P-content Form-A RNP, an RNA-P proteins complex virtually free from NP protein was isolated by cesium sulfate equilibrium centrifugation. The complex, containing only three P proteins (P1, P2, and P3), was still active in catalyzing RNA synthesis in vitro without addition of exogenous template, indicating that NP protein is not required for the catalysis of RNA synthesis. RNA synthesis by the isolated RNA-P proteins complex was dependent on either ApG or capped RNA primers, and required four ribonucleoside triphosphates as substrates. The RNA product in this reaction was hybridizable to viral RNA. A complex of one each of the three P proteins was separated from RNA by glycerol gradient centrifugation after ribonuclease treatment or cesium chloride equilibrium centrifugation.

DNA-Directed RNA Polymerases↗

PubMed2006

Discriminatory RNP remodeling by the DEAD-box protein DED1.

DExH/D proteins catalyze NTP-driven rearrangements of RNA and RNA-protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between a large number of RNA and RNP complexes in a physiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which a non-sequence specific DExH/D protein can discriminately remodel similar RNA complexes. We have measured in vitro the disassembly of model RNPs by two distinct DExH/D proteins, DED1 and NPH-II. Both enzymes displace the U1 snRNP from a tightly bound RNA in an active, ATP-dependent fashion. However, DED1 cannot actively displace the protein U1A from its binding site, whereas NPH-II can. The dissociation rate of U1A dictates the rate by which DED1 remodels RNA complexes with U1A bound. We further show that DED1 disassembles RNA complexes with slightly altered U1A binding sites at different rates, but only when U1A is bound to the RNA. These findings suggest that the "inability" to actively displace other proteins from RNA can provide non-sequence specific DExH/D proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. In addition, our study illuminates possible mechanisms for protein displacement by DExH/D proteins.

Adenosine Triphosphate↗