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Results for “RNP complex”

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PubMed1987

[Intranuclear actin microfilaments in the oocytes of the common frog].

For identification and distribution of actin microfilaments in hand-isolated nuclei of R. temporaria oocytes (stage 6, according to Dumont, 1972) different methods were used: heavy meromyosin decoration, antiactin immunofluorescence with monoclonal antibodies, staining with rhodamine phalloidin, and electrophoresis in polyacrylamide gel. The nuclei of R. temporaria oocytes contain a considerable quantities of actin microfilaments which form intranuclear meshwork. Microfilaments are connected with the nucleoli, nucleolar RNP-complexes and nuclear envelope. Immunofluorescence with antiactin monoclonal antibodies reveals a strong staining of microfilaments and nucleoli. A slight staining of nucleoli is observed after the treatment of nuclei with rhodamine phalloidin. A specific role of intranuclear microfilaments in direct transport of nucleolar material from the nucleus into the oocyte cytoplasm, in stabilization of the karyosphere (the late diplotene oocyte complex of chromosomes with numerous nucleoli) is discussed in addition to its keeping in a definite region of the nucleus. A supposition is drawn on the functional significance of the connection between microfilaments and nuclear matrix. Based on our own and literature data, a conclusion is drawn, that the intranuclear filament actin may be one of the leading components in morpho-functional organization of the nucleus as the whole.

Actin Cytoskeleton↗

PubMed1989

Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components?

By means of immunocytochemistry performed on cryosections of cultured cells, RNA polymerase I was localized mainly to nucleolar fibrillar centers. The labelling of nucleolar dense fibrillar components was low and depended on the cell type. In contrast, DNA topoisomerase I and RNP complexes containing U3 snRNA were enriched in dense fibrillar components, their occurrence in fibrillar centers being usually much less.

Adrenal Gland Neoplasms↗

PubMed1981

The translational inhibitor 10 S cytoplasmic ribonucleoprotein of chick embryonic muscle. Dissociation and reassociation.

Cytoplasmic 10S ribonucleoprotein (iRNP) is a potent inhibitor of mRNA translation in vitro and contains a 4 S translation inhibitory RNA species (iRNA) (Sarkar, S., Mukherjee, A. K., and Guha, C. (1981) J. Biol. Chem. 256, 5077-5086). This ribonucleoprotein has now been resolved into protein and RNA components by DEAE-cellulose chromatography in the absence of both K+ and Mg2+ ions. These cations are required for maintaining the nucleoprotein structure of iRNP. Incubation of the dissociated protein and RNA components in the presence of K+ and Mg2+ at 35 degrees C reconstitutes a 10 S particle which is indistinguishable from native iRNP with respect to the elution profile by gel filtration, UV spectra, buoyant density, resistance to pancreatic RNase, and ability to inhibit exogenous mRNA translation in vitro. Chick muscle tRNA and globin mRNA could not form an RNP complex with the protein moieties of iRNP. The separated proteins, unlike iRNA and iRNP, do not inhibit mRNA translation. Their function may be to protect iRNA from ribonuclease digestion, since iRNP is ribonuclease-resistant. The ability to dissociate the iRNP particle and to specifically reconstitute it from the separated components indicates that it is a unique cellular entity which is distinct from other ribonucleoproteins.

Animals↗

PubMed1993

Influencing the influenza virus: genetic analysis and engineering of the negative-sense RNA genome.

The genome of influenza virus has become amenable to genetic analysis as a result of newly developed methods for reconstitution of the ribonucleoprotein (RNP) complex. This allows a dissection of the regulatory signals and promoters required for transcription and replication of the viral genome. The ability to rescue chimeric viruses in vivo after transfection opens up a way to engineer viruses suitable for human immunization and carrying desirable antigenic determinants.

Base Sequence↗

PubMed1996

Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D.

Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processing. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc7O pre-mRNA intron. Competition studies indicate that a trans-acting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this factor is common to both intronic and non-intronic snoRNAs possessing nucleotide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region containing boxes C and D does not bind fibrillarin. Collectively, our results indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, thereby stabilizing the intronic snoRNA sequence in an RNP complex during processing.

Animals↗

PubMed1996

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.

Animals↗

PubMed1999

Mutation in neurofilament transgene implicates RNA processing in the pathogenesis of neurodegenerative disease.

A mouse neurofilament light subunit (NF-L) transgene with a 36 bp c-myc insert at the end of the coding region was found to have neuropathic effects on enteric and motor neurons of transgenic mice. The severity of phenotype was related directly to the levels of transgenic mRNA expression. High levels of transgene expression were lethal to newborn pups, causing profound alterations in the development of the enteric nervous system and extensive vacuolar changes in motor neurons. Lower levels of transgene expression led to a transient stunting of growth and focal alterations of enteric and motor neurons. Because the positioning of the c-myc insert coincided with the location of the major stability determinant of the NF-L mRNA (Cañete-Soler et al., 1998a,b), additional studies were undertaken. These studies showed that the c-myc insert alters the ribonucleoprotein (RNP) complexes that bind to the stability determinant and disrupts their ability to regulate the stability of the transcripts. The findings indicate that expression of an NF-L transgene with a mutant mRNA stability determinant is highly disruptive to enteric and motor neurons and implicate alterations in RNA processing in the pathogenesis of a neurodegenerative condition.

3' Untranslated Regions↗

PubMed2002

Complex formation between activator and RNA polymerase as the basis for transcriptional activation by MarA and SoxS in Escherichia coli.

Transcriptional activation in Escherichia coli is generally considered to proceed via the formation of an activator-DNA-RNA polymerase (RNP) ternary complex. Although the order of assembly of the three elements is thermodynamically irrelevant, a prevalent idea is that the activator-DNA complex is formed first, and recruitment of RNP to the binary complex occurs subsequently. We show here that the closely related activators, MarA, SoxS and Rob, which activate the same family of genes, are capable of forming complexes with RNP core or holoenzyme in the absence of DNA. In addition, we find that the ternary MarA-DNA-RNP and SoxS-DNA-RNP complexes are more stable than the corresponding Rob-DNA-RNP complex, although the binary Rob-DNA complex is often more stable than the corresponding MarA- or SoxS-DNA complexes. These results may help to explain certain puzzling aspects of the MarA/SoxS/Rob system. We suggest that activator-RNP complexes scan the chromosome and bind promoters of the regulon more efficiently than either RNP or the activators alone.

Bacterial Proteins↗

PubMed2003

An umbraviral protein, involved in long-distance RNA movement, binds viral RNA and forms unique, protective ribonucleoprotein complexes.

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.

Arachis↗

PubMed1999

Identification of RNA-protein contacts within functional ribonucleoprotein complexes by RNA site-specific labeling and UV crosslinking.

A variety of cellular processes are carried out by highly complex ribonucleoprotein (RNP) particles in which multiple RNA-RNA, RNA-protein, and protein-protein interactions occur. The spliceosome, which executes the nuclear pre-mRNA splicing reaction, is a particularly striking example of a complex RNP, containing a minimum of 50 distinct protein components as well as five small nuclear RNAs. In order to identify which among the numerous proteins may play critical roles in the splicing reaction, we have assembled spliceosomal complexes on pre-mRNA containing a single 32P-labeled nucleotide, isolated the complexes by gel filtration, and then carried out UV crosslinking. The combination of these three methods has allowed the identification of proteins that crosslink to critical sequence elements during each stage in spliceosome assembly. These methods should be generally applicable to the analysis of RNP complexes assembled in vitro.

Chromatography, Affinity↗

PubMed1984

Characterization of the effect of rimantadine on intracellular distribution and transport into the nuclei of infected cells of the parent influenza virus genome.

Rimantadine in the concentration of 50 micrograms/ml did not affect the distribution in subcellular fractions of MDCK cells of the parent influenza virus A/Khabarovsk/74/77(H1N1) labelled with 14C-amino acids, 3H-nucleosides or 32P-orthophosphoric acid. In the presence of rimantadine viral structures of heterogenous composition were detected in the nuclei of infected cells. Their buoyant densities of 1.34-1.36, 1.41-1.42 and 1.28-1.32 g/cm3 in the CsCl gradient were characteristic for RNP and "nucleoids" (complex of RNP and M protein), respectively. Similar structures were also detected in the nuclei of cells infected in the absence of rimantadine. No primary transcription of the viral genome was registered in the presence of rimantadine, although the genetic material in the nuclei was present in a form suitable for transcription.

Adamantane↗

PubMed1985

The nuclear binding of estradiol stimulates ribonucleoprotein transport in the rat uterus.

An inverse relationship exists in the rat uterus between the endogenous estradiol concentration and the rate of accumulation within the nuclei of ribonucleoproteins (RNP) that bind estradiol. Exposure of the uterine nuclei to physiological concentrations of estradiol, either in vivo or in vitro, results in the hormone binding to the nuclear RNP and the transport of the RNP-estradiol complex from the nuclei. Evidences suggest that the RNP-estradiol complex, thus released upon in vivo exposure of the nuclei to estradiol, associates with the ribosomes forming polysomes. The polysome profiles show that the hormone-binding activity is mainly associated with the rapidly sedimenting polysomal fractions.

Adenosine Triphosphate↗

PubMed2024

Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.

Gene Editing↗

PubMed1987

An immunoassay differentiating sera with antibodies to Sm alone, antibodies to Sm/RNP complex, and antibodies to RNP alone.

Combined DEAE-Sephacel and hydroxyapatite chromatography resulted in a separation of free Sm antigen from the Sm/RNP complex in rabbit thymus extracts. In Western blots, the free Sm preparation contained an immunoreactive 14-kd (D) protein, whereas the Sm/RNP complex contained, in addition to the 14-kd protein, a 68-kd-reactive protein and its putative degradation fragments. Ethidium bromide staining of these preparations separated by agarose gel electrophoresis showed that the Sm/RNP preparation contained RNA, but the free Sm preparation did not. These preparations were used as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera characterized as anti-Sm only, anti-Sm/RNP, and anti-RNP only were assayed. A quotient (Q) was derived from the ELISA optical density obtained with Sm/RNP as antigen divided by the optical density obtained with free Sm as antigen. Q values less than 4.0 characterized sera with anti-Sm only, values of 4.0-12.0 were observed in sera with anti-Sm plus anti-RNP, and values greater than 12.0 in sera with anti-RNP only. Sera with antibodies to other nuclear antigens were not reactive in this system. It has traditionally been difficult to identify sera with anti-RNP when this is present simultaneously with, and in lower concentration than, anti-Sm. With this method, such sera can be identified by their Q value, which falls in an intermediate region between a lower Q for anti-Sm and a higher Q for anti-RNP.

Autoantibodies↗

PubMed1977

[Nuclear bodies of lymphocytes in the lymph nodes of guinea pigs treated with phytohemagglutinin in vivo].

Nuclear bodies (NB) were studied in guinea pigs regional lymph node cells after a single subcutaneous application of Phytohaemagglutinin Difco (PHA). A distinct reaction of the lymph node cells was established on the 3rd, 6th and 9th days consisting of increased number of lymphoblasts and large basophilic cells (LBC). Ultrastructural descriptive and morphometric methods were used. Authors revealed nuclear bodies to be present mostly in activated lymph node cells (lymphoblasts and LBC). Predominance of NB having RNP particles (complex type NB) was found. Simple NB (without RNP particles) had been seen rarely in small (resting lymphocytes. It was established NB to be larger, but leis im member. It is discussed on the participation of NB (Complex type) in the nuclear part of the protein synthesis.

Animals↗

PubMed2000

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed.

Base Sequence↗

PubMed2005

Lymphotropic Herpesvirus saimiri uses the SMN complex to assemble Sm cores on its small RNAs.

The lymphotropic Herpesvirus saimiri (HVS) causes acute leukemia, T-cell lymphoma, and death in New World monkeys. HVS encodes seven small RNAs (HSURs) of unknown function. The HSURs acquire host Sm proteins and assemble Sm cores similar to those found on the spliceosomal small nuclear RNPs (snRNPs). Here we show that, like host snRNPs, HSURs use the SMN (survival of motor neurons) complex to assemble Sm cores. The HSURs bind the SMN complex directly and with very high affinity, similar to or higher than that of host snRNAs, and can outcompete host snRNAs for SMN-dependent assembly into RNPs. These observations highlight the general utility of the SMN complex for RNP assembly and suggest that infectious agents that engage the SMN complex may burden SMN-dependent pathways, possibly leading to a deleterious reduction in available SMN complex for essential host functions.

Animals↗

PubMedDate not supplied

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. III. The DNA-binding activity of nuclear alpha-RNP. The Signal Transmission from Growth Factors Group of the Medical Faculty, F. Schiller University, Jena, Germany].

DNA-binding activity of small nuclear alpha-RNP identified in acid-soluble fraction of chromatin of human proerythroleukemic cell line K-562 was studied using the technique of gel retardation. We found that nuclear alpha-RNP isolated from K-562 cells through treatment with dimethylsulfoxide, an agent inducing differentiation, acquire a capacity to specific interaction with Alu repeats of DNA leading to the formation of alpha-RNP-Alu-DNA complexes; nuclear alpha-RNP from cells that were not treated with dimethylsulfoxide do not show such capacity, although they are tightly bound with chromatin in the cell. Thus, the capacity of nuclear alpha-RNP to direct interaction with DNA Alu repeats appearing after the induction of K-562 cells to differentiation along erythroid pathway is an inducible property. We discuss hypothesis about the involvement of nuclear alpha-RNP in the control of expression of inducible genes at the level of chromatin and interaction with DNA.

Base Sequence↗