RNP Particles Research Articles

A study of the sedimentation properties of the U-snRNPs of mouse embryoid bodies (OTT6050) by sucrose gradient centrifugation under physiological ionic conditions and electrophoretic analysis of the RNA in various fractions.

Fractionation of nuclear extracts of mouse embryoid bodies (EBs; OTT6050) on sucrose gradients (5-20%) under conditions of physiological ionic strength (150 mM NaCl, 1mM MgCl2) and an analysis of the RNA in various fractions by gel electrophoresis revealed that the major U small nuclear ribonucleoproteins (U-snRNPs; U1a, U1b, U2, U4, U5 and U6-snRNPs) sediment over a wide region of the gradient, although they sediment preponderantly in the light region of the gradient. This result suggests that, under these experimental conditions, some of the populations of snRNPs exist as free particles separated from large nuclear RNP particles, while some of the populations associate with them in EBs. Furthermore, all species of these major U-snRNPs appear to associate with the larger nuclear RNP particles of EBs, since all these species sediment in the heavier fractions (approximate greater than or equal to 60S) of the 15-40%/50% gradients. The relative abundance of the various species of major U-snRNPs can also be observed to vary among the fractions of the gradients. A similar analysis of the post-mitochondrial cytoplasmic fraction showed that some leakage of the major U-snRNPs, but not the selective leakage of any particular species of U-snRNP, from the nuclear fraction, occurred during aqueous fractionation of the cells. Some species of RNA, larger than the snRNAs U1a/b and U2 respectively were also detected in the cytoplasmic fraction.

Crosslinking of transcription factor TFIIIA to ribosomal 5S RNA from X. laevis by trans-diamminedichloroplatinum (II).

Trans-diamminnedichloroplatinum (II) was used to induce reversible crosslinks between 5S rRNA and TFIIIA within the 7S RNP particle from X. laevis immature oocyte. The crosslinked fragments have been unambiguously identified. These fragments exclusively arise from three RNA regions centered around the hinge region at the junction of the three helical domains. Major crosslinking sites are located in region 9-21 (comprising loops A and helix II) and region 54-71 (comprising loop B, helices II and V). A minor site is also found in the 3' part of helix I and helix V (region 100-120). Our results point to the crucial role of the junction region and of the three-dimensional folding of the RNA in the recognition of the 5S rRNA by TFIIIA.

Prosomes, small cytoplasmic RNP particles, contain glycoproteins

Prosomes, ubiquitous small ribonucleoprotein complexes, were isolated from the cytoplasm of erythropoietic mouse cells induced by Friend leucemia virus. We present evidence that some of the prosomal proteins are glycosylated. Specific reactions with the biotinylated lectins concanavalin agglutinin (Con A), Solanum tuberosum agglutinin (STA) and Limulus polyphemus agglutinin (LPA) indicate that the carbohydrate moieties contain N-acetylneuraminic acid, N-acetylglucosamine and mannosyl- or glucosyl-residues. Glycosylation of prosomal proteins could explain the resistance of prosomes to proteinase K digestion.

Synthesis and Transport of a Specific Premessenger RNP Particle

Abstract The three dimensional structure of a specific premessenger RNP particle has been studied using electron microscope tomography. The structure of the globular particle in the nuclear sap can be described as a bent ribbbon with the ends in close proximity to each other. During translocation the ribbon opens up and elongates as it passes through the nuclear pore.

Anterior determinants in embryos of Chironomus samoensis: characterization by rescue bioassay

Embryos of Chironomus samoensis are programmed, by anterior u.v. irradiation, to form the abnormal body pattern 'double abdomen'. Most double abdomen embryos show a mirror-image duplication of abdominal segments in the absence of cephalic or thoracic segments. Such embryos can be 'rescued', i.e. restored to normal development, by microinjection of cytoplasm or RNA from unirradiated donor embryos. Most of the rescued embryos look completely normal and many of them hatch spontaneously. The rescuing activity decreases from the anterior to the posterior pole in the donor cytoplasm and must be delivered near the anterior pole of the recipient for maximum efficiency. Rescuing activity is present in total RNA extracted from whole, unirradiated embryos. Upon fractionation, the activity is associated with poly(A)+ RNA, with LiCl precipitate depleted of RNA smaller than 250 nucleotides (nt) and with a sucrose gradient fraction depleted of RNA larger than 500 nt. Corresponding fractions of RNA from Xenopus oocytes have no rescuing activity. The activity of Chironomus RNA is sensitive to u.v. irradiation with low fluence affecting less than 2% of the pyrimidine bases. Rescuing activity is present in cytoplasm until the blastoderm stage but disappears earlier from poly(A)+ RNA. Rescuing activity is also present, and localized, in cytoplasm of embryos from two related dipterans, Smittia sp. and Drosophila melanogaster, although the extent of rescue observed in Chironomus decreases with the phylogenetic distance between donor and recipient. The results of these and previous experiments indicate that dipteran embryos contain localized RNP particles acting as anterior determinants. In Chironomus, the activity of these particles seems to depend on the integrity of polyadenylated RNA of about 250 to 500 nt length.

Structure and transport of a specific premessenger RNP particle

Structure and transport of a specific premessenger RNP particle

Protein kinase activity associated with stored messenger ribonucleoprotein particles of Xenopus oocytes.

As the oocytes of Xenopus laevis grow and develop they accumulate vast stores of mRNA for use during early embryogenesis. The stored mRNA is stabilized and may be prevented from being translated in oocytes by the binding of a defined set of oocyte-specific proteins to form messenger RNP (mRNP) particles. A key event in the interaction of protein with mRNA is the phosphorylation of those few polypeptides that bind directly to all classes of polyadenylated mRNA. In this study we show that the phosphorylating enzyme (protein kinase), in addition to its target phosphoproteins, is an integral component of the mRNP particles. This association extends through various stages in the formation and use of the mRNP particles. Examination of material from oocytes of an early developmental stage (early stage 1), when the level of accumulated mRNA is low, reveals an excess of protein particles free of RNA, sedimenting at 6-18 S, and containing protein kinase activity and mRNA-binding phosphoproteins. At stages of maximum rate of mRNA accumulation (stages 1 and 2), the phosphoproteins and kinase are found primarily in individual mRNP particles that sediment at 40-80 S. As ribosomes become abundant (stages 2 and 3), the mRNP particles tend to interact with ribosomal subunits, at least in vitro, to form blocked translation initiation complexes that sediment at 80-110 S. These results are compared with observation on stored mRNP in other developmental systems.

A new type of prosome‐like particle, composed of small cytoplasmic RNA and multimers of a 21‐kDa protein, inhibits protein synthesisin vitro

A large fraction of the translationally repressed non-globin messenger RNA in duck erythroblasts is present in non-polyribosomal free mRNP structures which sediment in the 30-40-S range ('35 S'). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.-P. Schmid et al. (1984) EMBO J. 3, 29-34]. Buoyant density measurements and chromatography on oligo(dT)-cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS. This prosome-like particle inhibits the translation of mRNA in vitro. It is composed primarily of multimers of a single 21-kDa protein and at least one species of RNA of about 80-100 nucleotides. It is resistant to dissociation by 2 M CS2SO4 and 1% SDS; the 21-kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)-rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA. These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome-like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously identified.

Instability of the anteroposterior axis in spontaneous double abdomen (sda), a genetic variant of Chironomus samoensis (Diptera, Chironomidae)

ABSTRACT We have isolated a laboratory strain of Chironomus samoensis in which determination of the anteroposterior egg polarity is disturbed. Most conspicuous is the spontaneous formation of ‘double abdomen’ embryos where head and thorax are replaced by a mirror image of the abdomen. Such double abdomens are found in about half of the egg clusters in this strain, which we call the spontaneous double abdomen (sda) strain as opposed to the normal (N) strain. Also observed in the sda strain, although less frequently, are ‘double cephalon’ embryos showing a mirrorimage duplication of cephalic segments in the absence of thorax and abdomen. Moreover, embryos from the sda strain tend to form cells at the anterior pole resembling the pole cells at the posterior pole. Reciprocal crossings between the sda and the N strain indicate that the sda trait is inherited maternally. Spontaneous double abdomen formation is correlated with signs of disturbed egg architecture, including extruded yolk and detached cells. Double cephalons can also be generated by centrifuging embryos from the N strain, whereas centrifugation of sda embryos produces mostly double abdomens. Double abdomen formation can be induced experimentally by anterior u.v. irradiation of embryos from either strain. The sda trait and u.v. irradiation act in a synergistic fashion. The data suggest that the sda trait may be caused by one or more genomic mutations interfering indirectly with the activity of anterior determinants, i.e. cytoplasmic RNP particles necessary for the development of anterior segments. The sda defects may be ascribed to alterations in cytoskeletal components involved in anchoring anterior determinants and segregating them into anterior blastoderm cells.

Monoclonal autoantibody from a (new zealand black x new zealand white)F1 mouse and some human scleroderma sera target an MTr 34,000 nucleolar protein of the U3 RNP particle

A monoclonal IgG2a antinucleolar autoantibody (72B9) was obtained by fusion of spleen cells from a (New Zealand black x New Zealand white)F1 mouse with myeloma cells (P3x63Ag8.653). Antibody 72B9 recognized a highly conserved nucleolar antigen present in both animal and plant cells. The staining pattern produced by antibody 72B9 in different cell substrates was identical with those obtained by scleroderma antibodies reactive with a basic (pI 8.5) nucleolar protein of Mr 34,000, which is associated with the U3 RNP particle. Western blotting further confirmed its reactivity with this scleroderma-related U3 RNP protein.

Identification of protein antigens associated with the nuclear matrix and with clusters of interchromatin granules in both interphase and mitotic cells.

Monoclonal antibody 3C5 recognizes a family of protein antigens present predominantly within the nucleus of interphase cells. We have shown previously that the epitope recognized by 3C5 is phosphorylated and have concluded that the proteins defined by this antibody share a common phosphorylation site. Using a combination of immunofluorescence microscopy and immunogold labelling in conjunction with electron microscopy, we have studied the distribution of 3C5-reactive material within interphase and mitotic cells. Antibody 3C5 was found to label specific structures within the interphase nucleus that, on the basis of their characteristic granulofibrillar morphology and strong staining with bismuth, have been identified as clusters of interchromatin granules (IG clusters). Double-labelling experiments with 3C5 and monoclonal antibodies to DNA have shown that these structures contain no detectable DNA. However, by indirect immunofluorescence, we have shown that 3C5-reactive nuclear structures do label with human autoantibodies to the Sm antigen, a component of small nuclear RNP particles (snRNPs). Granulofibrillar structures that stained strongly with bismuth, and were morphologically identical to nuclear IG clusters, were observed in the cytoplasm of mitotic cells. These structures also labelled with 3C5 but not with anti-Sm antibodies. Our results suggest that IG clusters remain essentially intact through mitosis though some snRNP components are apparently lost. In situ extraction of cultured cells with Triton X-100, micrococcal nuclease and 1-2M-NaCl failed to deplete 3C5-reactive material in either interphase or mitotic cells, though some redistribution was evident. In addition, 3C5-reactive proteins were identified in nuclear matrices prepared from rat liver by high-salt extraction procedures. However, the recovery of such proteins was strongly influenced by the preparation technique employed. Our results suggest that 3C5-reactive proteins and IG clusters are anchored to, but not integral components of, salt-resistant structural elements of the interphase nucleus and the mitotic cytoplasm, presumably the nuclear matrix and the cytoskeleton, respectively.

Modifications of the chromatin arrangement induced by ethidium bromide in isolated nuclei, analyzed by electron microscopy and flow cytometry.

Ethidium bromide (EB) is widely used for investigating the DNA conformation in chromatin both with conventional and cytofluorimetric techniques. Since the interaction of the dye with DNA should result in structural deformations which can be different in isolated or in situ chromatin, a study has been performed on the effects caused by different amounts of EB and the analogous propidium iodide on isolated nuclei, in which chromatin maintains its native relationships with the other nuclear structures (envelope, nucleolus, interchromatin RNP, nuclear matrix). The results obtained by comparing ultrastructural observations in thin sections and in freeze-fracturing with conformational analysis in multiparameter flow cytometry indicate that the phenanthridinic fluorochromes, especially at the high concentrations used for cytofluorimetric analyses, cause deep rearrangements of the chromatin in situ. These effects consist both in aggregation and condensation of the fibers into the dense chromatin domains, and in an increase of the supernucleosomal configuration associated with an enlargement of interchromatin spaces in which the RNP particles appear particularly evident. These results, discussed with those available on isolated chromatin, suggest that any unwinding effect of the intercalating dyes on the DNA cause a general condensation of chromatin as a consequence of the constraints which characterize the organization of the chromatin inside the nucleus.

Cell cycle-dependent changes in conformation and composition of nucleosomes containing human histone gene sequences.

Unfolding of the nucleosomes in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3 and permits the selective recovery of the unfolded nucleosomes by mercury-affinity chromatography. This new technique has been used to compare the nucleosomal proteins and their postsynthetic modifications in the unfolded and the compactly beaded nucleosomes of HeLa cells in logarithmic growth, and at different stages of the growth cycle. The Hg-bound nucleosomes are shown to be deficient in replicating DNA sequences, but to remain associated with fragments of nascent RNA chains (or RNP particles) during gradient centrifugations. Both nucleosome fractions contain a full complement of "core" histones but differ with respect to postsynthetic modifications. The Hg-bound nucleosomes contain high levels of the tri- and tetra-acetylated forms of histones H3 and H4. The unbound nucleosomes are deficient in acetylated histones but enriched in phosphorylated H2A. In synchronized HeLa cells, histone H2A and H4 gene sequences occur in the Hg-bound nucleosomes during the S-phase when their transcription takes place, but not in the G2-phase when the genes are repressed.

Structural and biochemical characterization of a specific premessenger RNP particle

Structural and biochemical characterization of a specific premessenger RNP particle

Monoclonal antibodies against large nuclear RNP particles

Monoclonal antibodies against large nuclear RNP particles

Reconstitution and protein-RNA mapping of the human U1 small nuclear RNP particle

Reconstitution and protein-RNA mapping of the human U1 small nuclear RNP particle

Distribution of the 70K U1 RNA-associated protein during interphase and mitosis. Correlation with other U RNP particles and proteins of the nuclear matrix.

Earlier studies suggested that the 70K (70 X 10(3) Mr) polypeptide is a nuclear matrix (associated) protein since it is the only U1 RNP-associated antigen that is not released from the nucleus after treatment of the cell with, successively, detergents, DNase I and/or RNase A and high salt. The possibility that the 70K protein functions in the binding of U1 RNP to the nuclear matrix is now further substantiated by the finding that U1 RNP particles that did or did not contain the 70K protein could be isolated, depending on the method of isolation. When U1 RNP particles were obtained by means of sonic disruption of the nucleus they contained the 70K polypeptide, whereas particles that were isolated by extraction at room temperature and a slightly alkaline pH lacked the 70K protein but contained the intact U1 RNA and the other U1 RNA-associated proteins. During interphase the localization of the 70K protein is restricted to the nucleus, giving a dot-like distribution pattern with exclusion of the nucleoli. During prophase to late anaphase the protein is dispersed throughout the entire cytoplasm with the exception of the chromatin regions. Immunofluorescence studies, using a monoclonal anti-70K antibody in combination with human autoimmune sera that react with U1 RNA-associated proteins, demonstrate that the 70K protein is localized in those areas of the cell where other U RNP proteins occur, also during mitosis. Topoisomerase I and nuclear lamins, typical nuclear matrix proteins, show completely different distribution patterns in all phases of the cell cycle. Assembly of the nuclear envelope is attended by the re-formation of the clustered appearance of the 70K antigen. These results suggest that, although associated with the nuclear matrix fraction in interphase cells, the 70K protein remains associated with the U1 RNP particles during cell division.

Three-dimensional structure of a specific pre-messenger RNP particle established by electron microscope tomography.

Electron microscope tomography has been used to refine the structural analysis of individual RNP particles synthesized on Balbiani ring genes in Chironomus tentans. The spherical particles have a diameter of 500 A and are composed of a thick RNP ribbon bent into an asymmetrical, four-domain, ring-like configuration. The first domain, containing the 5' end of the transcript, and the fourth domain, containing the 3' end, are close to each other.

Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.

Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei

The response of isolated rat liver and murine erythroleukemia nuclei to phospholipid liposomes has been monitored with different techniques, by studying the endogenous RNA synthesis, the release of transcripts in the medium, the pattern of acid-extractable nuclear proteins and the ultrastructural morphology. Total transcription in rat liver and β-globin mRNA synthesis in MEL nuclei are increased by PS and reduced by PC. These changes of RNA polymerase activity, and the transport of RNAs from nucleus as well as the nuclear protein changes, correlate with structural transitions which occur in both types of nuclei, consisting of euchromatization with loss of RNP particles in the case of PS and opposite effects with PC. The significance of these modifications in relationship to the possible involvement of phospholipids in the control of gene expression is discussed.