scispace - formally typeset
Search or ask a question

Showing papers on "Nucleolus published in 1994"


Journal ArticleDOI
TL;DR: Immunofluorescence and immunogold electron microscopy with NAP57 specific antibodies show colocalization with Nopp140 to the dense fibrillar component of the nucleolus, to coiled bodies, and to the nucleoplasm, which suggest that Nopp 140 and NAP 57 are indeed associated with each other in these nuclear structures.
Abstract: We report the identification and molecular characterization of a novel nucleolar protein of rat liver. As shown by coimmunoprecipitation this protein is associated with a previously identified nucleolar protein, Nopp140, in an apparently stoichiometric complex and has therefore been termed NAP57 (Nopp140-associated protein of 57 kD). Immunofluorescence and immunogold electron microscopy with NAP57 specific antibodies show colocalization with Nopp140 to the dense fibrillar component of the nucleolus, to coiled bodies, and to the nucleoplasm. Immunogold staining in the nucleoplasm is occasionally seen in the form of curvilinear tracks between the nucleolus and the nuclear envelope, similar to those previously reported for Nopp140. These data suggest that Nopp140 and NAP57 are indeed associated with each other in these nuclear structures. The cDNA deduced primary structure of NAP57 shows a protein of a calculated molecular mass of 52,070 that contains a putative nuclear localization signal near its amino and carboxy terminus and a hydrophobic amino acid repeat motif extending across 84 residues. Like Nopp140, NAP57 lacks any of the known consensus sequences for RNA binding which are characteristic for many nucleolar proteins. Data bank searches revealed that NAP57 is a highly conserved protein. A putative yeast (S. cerevisiae) homolog is 71% identical. Most strikingly, there also appears to be a smaller prokaryotic (E. coli and B. subtilis) homolog that is nearly 50% identical to NAP57. This indicates that NAP57 and its putative homologs might serve a highly conserved function in both pro- and eukaryotes such as chaperoning of ribosomal proteins and/or of preribosome assembly.

251 citations


Journal ArticleDOI
TL;DR: After dispersing the granular component and the dense fibrillar component by a hypotonic treatment, removal of most chromatin and preparation of resinless sections, fibrillsar centres remained fixed to a nucleoskeleton and are incorporated into a model for rRNA transcription.
Abstract: Sites of transcription of ribosomal RNA in HeLa cells were visualized by electron microscopy. Cells were either incubated with Br-uridine, or permeabilized and then incubated with BrUTP, before sites containing Br-RNA were immunolabeled with gold particles. Short incubations ensured that most incorporated analogue remained at synthetic sites. Fibrillar centres were unlabelled except at their periphery; label was concentrated over certain regions of the surrounding dense fibrillar component. These results suggest that the dense fibrillar component is the site of rRNA transcription. After dispersing the granular component and the dense fibrillar component by a hypotonic treatment, removal of most chromatin and preparation of resinless sections, fibrillar centres remained fixed to a nucleoskeleton. These structural and functional features are incorporated into a model for rRNA transcription.

212 citations


Journal ArticleDOI
TL;DR: Results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis, similar to those reported here for the localization of U3 snRNA.
Abstract: We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, containing factors essential for pre-rRNA processing, are recruited to these sites of rRNA transcription and processing.

159 citations


Journal ArticleDOI
TL;DR: It is demonstrated that the prognostic test for human cancer cell proliferation is largely based on the amount of the nucleolar proteins, nucleolin, and protein B23, which are not directly involved in ribosomal gene transcription.

157 citations


Journal ArticleDOI
TL;DR: It is reported that a 15-kDa cellular protein called EAP (for EBER associated protein), previously shown to bind EBER1, is in fact the ribosomal protein L22, and in situ hybridization indicates that the EBER RNPs are predominantly nucleoplasmic, suggesting that L22 relocalization correlates with binding to Eber1 in vivo.
Abstract: Epstein-Barr virus (EBV), an oncogenic herpesvirus, encodes two small RNAs (EBERs) that are expressed at high levels during latent transformation of human B lymphocytes Here we report that a 15-kDa cellular protein called EAP (for EBER associated protein), previously shown to bind EBER1, is in fact the ribosomal protein L22 Approximately half of the L22 in EBV-positive cells is contained within the EBER1 ribonucleoprotein (RNP) particle, whereas the other half residues in monoribosomes and polysomes Immunofluorescence with anti-L22 antibodies demonstrates that L22 is localized in the cytoplasm and the nucleoli of uninfected human cells, as expected, whereas EBV-positive lymphocytes also show strong nucleoplasmic staining In situ hybridization indicates that the EBER RNPs are predominantly nucleoplasmic, suggesting that L22 relocalization correlates with binding to EBER1 in vivo Since incubation of uninfected cell extracts with excess EBER1 RNA does not remove L22 from preexisting ribosomes, in vivo binding of L22 by EBER1 may precede ribosome assembly The gene encoding L22 has recently been identified as the target of a chromosomal translocation in certain patients with leukemia, suggesting that L22 levels may be a determinant in cell transformation

156 citations


Journal ArticleDOI
TL;DR: The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.
Abstract: A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.

139 citations


Journal ArticleDOI
TL;DR: The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly.
Abstract: A complex structure, visible by electron microscopy, surrounds each chromosome during mitosis. The organization of this structure is distinct from that of the chromosomes and the cytoplasm. It forms a perichromosomal layer that can be isolated together with the chromosomes. This layer covers the chromosomes except in centromeric regions. The perichromosomal layer includes nuclear and nucleolar proteins as well as ribonucleoproteins (RNPs). The list of proteins and RNAs identified includes nuclear matrix proteins (perichromin, peripherin), nucleolar proteins (perichro-monucleolin, Ki-67 antigen, B23 protein, fibrillarin, p103, p52), ribosomal proteins (S1) and snRNAs (U3 RNAs). Only limited information is available about how and when the perichromosomal layer is formed. During early prophase, the proteins extend from the nucleoli towards the periphery of the nucleus. Thin cordon-like structures reach the nuclear envelope delimiting areas in which chromosomes condense. At telophase, the proteins are associated with the part of the chromosomes remaining condensed and accumulate in newly formed nucleoli in regions where chromatin is already decondensed. The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly.

139 citations


Journal ArticleDOI
TL;DR: It is demonstrated that Srp1p is essential for maintenance of the crescent-shaped nucleolar structure, RNA transcription, and the proper functions of microtubules as inferred from analysis of nuclear division/segregation and immunofluorescence microscopy of micro Tubules.
Abstract: SRP1, a suppressor of certain temperature-sensitive mutations in RNA polymerase I in Saccharomyces cerevisiae, encodes a protein that is associated with nuclear pores. By using a system of conditional SRP1 expression and by isolating temperature-sensitive srp1 mutants, we have demonstrated that Srp1p is essential for maintenance of the crescent-shaped nucleolar structure, RNA transcription, and the proper functions of microtubules as inferred from analysis of nuclear division/segregation and immunofluorescence microscopy of microtubules. Different mutant alleles showed significantly different phenotypes in relation to these apparently multiple functional roles of the protein. We have also found that eight imperfect 42-amino-acid tandem repeats present in Srp1p are similar to the 42-amino-acid repeats in armadillo/plakoglobin/beta-catenin proteins present in adhesive junction complexes of higher eukaryotes. We discuss this similarity in connection with the observed pleiotropic effects of srp1 mutations.

130 citations


Journal ArticleDOI
TL;DR: The subcellular localization of PAB II was investigated with an antibody affinity-purified from rabbit serum raised against the purified protein, and the distribution corresponds largely to that of other factors involved in the processing of pre-mRNA and is thus in agreement with the proposed role of the protein in polyadenylation.

128 citations


Journal ArticleDOI
TL;DR: This is the first report of coiled bodies and Sm staining in the nucleolus of mammalian cells in a number of different breast cancer cell lines.
Abstract: Coiled bodies are a special type of small round nuclear body, composed of coiled fibers and granules, especially prominent in the nucleoplasm of highly active cells (Brasch and Ochs (1992) Exp. Cell Res. 202, 211-223). Although no specific function has been assigned to coiled bodies, they contain spliceosome snRNAs and proteins, as well as the nucleolar U3 RNA-associated protein fibrillarin. In the present study, we have used antibodies to the coiled body-specific protein p80-coilin, together with double-label immunofluorescence, confocal microscopy and immunoelectron microscopy, to examine the distribution of coiled bodies in a number of different breast cancer cell lines. By immunofluorescence, all cell lines had prominent coiled bodies in the nucleoplasm and several cell lines appeared to have coiled bodies within the nucleolus itself. Double-label immunofluorescence and confocal laser scanning microscopy confirmed the nucleolar localization of coiled bodies. Besides containing p80-coilin, nucleoplasmic and nucleolar coiled bodies contained fibrillarin and Sm proteins. By conventional and immunoelectron microscopy, nucleolar coiled bodies appeared as discrete structures within the nucleolus in a number of different morphotypes, distinct from the normal nucleolar domains of granular component, dense fibrillar component, and fibrillar centers. While the significance of finding coiled bodies in the nucleolus of certain breast cancer cell lines is at present unknown, this represents the first report of coiled bodies and Sm staining in the nucleolus of mammalian cells.

119 citations


Journal ArticleDOI
TL;DR: The oligomeric small heat-shock protein hsp27, also denoted hsp28, is constitutively expressed in several mammalian cells and displays a phosphorylation status that is related to cellular growth and differentiation.
Abstract: The oligomeric small heat-shock protein hsp27, also denoted hsp28, is constitutively expressed in several mammalian cells and displays a phosphorylation status that is related to cellular growth and differentiation. This protein is related to α-crystallin and has strong sequence similarity with an in vitro inhibitor of actin polymerization. Here, we have analyzed hsp27 phosphorylation, cellular localization and structural organization following serum stimulation of serum-starved HeLa cells. hsp27 is dephosphorylated in starved cells and quantitatively recovered in the form of small structures (<200 kDa) present in the soluble phase of the cytoplasm. Immediately after the addition of serum to starved cells, a rapid phosphorylation and complex changes in the intracellular distribution and structural organization of hsp27 are observed. Phosphorylation essentially occurs at the level of small hsp27 structures (<200 kDa) and is concomitant with the increased molecular mass (up to 700 kDa) of a fraction of this protein. Serum treatment also induced the detergent-sensitive association of another fraction of hsp27, still in the form of small and dephosphorylated structures, with cellular particulate fractions. Contrasting with these observations, hsp70 had the tendency to concentrate into nucleoli during serum starvation.

Journal ArticleDOI
TL;DR: The deletion of the sequence complementary to U3 in the ETS mimics all the known effects of the depletion of U2 in trans and indicates that an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA, is identified.
Abstract: The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.

Journal ArticleDOI
TL;DR: 31RRRGL35 is a nuclear localization signal responsible for the nucleolar targeting of human angiogenin and rapidly imported into the nucleus and localized to the nucleolus, whereas the "mutant" peptide is not.

Journal ArticleDOI
TL;DR: It is proposed that NOP77p mediates specific interactions between NOP1p and the pre‐rRNA and contains three canonical RNA recognition motifs (RRMs), suggesting that it is an RNA binding protein.
Abstract: The nucleolar protein fibrillarin (encoded by the NOP1 gene in yeast), is required for many post-transcriptional steps in yeast ribosome synthesis A screen for mutations showing synthetic lethality with a temperature sensitive nop1-5 allele led to the identification of the NOP77 gene NOP77 is essential for viability and encodes a nucleolar protein with a predicted molecular weight of 77 kDa Depletion of NOP77p impairs both the processing and methylation of the pre-rRNA The processing defect is greatest for the pathway leading to 25S rRNA synthesis, and is distinctly different from that observed for mutations in other nucleolar components NOP77p contains three canonical RNA recognition motifs (RRMs), suggesting that it is an RNA binding protein The NOP77 allele which complements the synthetic lethal nop1 strains has an alanine at position 308, predicted to lie in helix alpha 1 of RRM3, whereas the non-complementing nop77-1 allele contains a proline at the corresponding position We propose that NOP77p mediates specific interactions between NOP1p and the pre-rRNA

01 Jan 1994
TL;DR: In this paper, the distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labeled oligonucleotide probes.
Abstract: The distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labelled oligonucleotide probes. The location of the hybrids by immunofluorescence microscopy and at the ultrastructural level was correlated with the distribution of two nucleolar proteins, nucleolin and fibrillarin. The U3 snRNA molecules persist throughout mitosis in close association with the nucleolar remnant. U3 snRNA is present in the prenucleolar bodies (PNBs) and could participate in nucleologenesis in association with several nucleolar proteins such as nucleolin and fibrillarin. The interaction of U3 snRNP with the 5′ external spacer of pre-RNA newly synthesized by active NORs is proposed to be the promoting event of nucleologenesis.

Journal ArticleDOI
01 Oct 1994-Virology
TL;DR: It was demonstrated that, while extensive redistribution of Rev was still capable of inducing expression of HIV structural gene expression under these conditions, Rev activity does not appear to be dependent on either an intact nucleolus or the accumulation of the protein in the nucleus.

Journal ArticleDOI
TL;DR: The interaction of U3 snRNP with the 5' external spacer of pre-RNA newly synthesized by active NORs is proposed to be the promoting event of nucleologenesis.
Abstract: The distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labelled oligonucleotide probes. The location of the hybrids by immunofluorescence microscopy and at the ultrastructural level was correlated with the distribution of two nucleolar proteins, nucleolin and fibrillarin. The U3 snRNA molecules persist throughout mitosis in close association with the nucleolar remnant. U3 snRNA is present in the prenucleolar bodies (PNBs) and could participate in nucleologenesis in association with several nucleolar proteins such as nucleolin and fibrillarin. The interaction of U3 snRNP with the 5' external spacer of pre-RNA newly synthesized by active NORs is proposed to be the promoting event of nucleologenesis.

Journal ArticleDOI
TL;DR: Roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure are suggested.
Abstract: We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.

Journal ArticleDOI
TL;DR: The possible functions of MTR2 and the yeast nucleolus in mRNA export are discussed and it is suggested that the disorganization of theucleolus thus depends on mRNA accumulation in these mutants.
Abstract: We have identified a set of genes that affect mRNA transport (mtr) from the nucleus to the cytoplasm of Saccharomyces cerevisiae. One of these genes, MTR2, has been cloned and shown to encode a novel 21-kDa nuclear protein that is essential for vegetative growth. MTR2 shows limited homology to a protein implicated in plasmid DNA transfer in Escherichia coli. PolyA+RNA accumulates within the nucleus of mtr2-1 in two to three foci at 37 degrees C. mRNA, tRNA, and rRNA synthesis continue as do pre-mRNA splicing, tRNA processing, and rRNA export at 37 degrees C. Under these conditions the polyA tail length increases, and protein synthesis is progressively inhibited. Nucleolar antigens also redistribute to two to three nuclear foci at 37 degrees C, and this redistribution depends on ongoing transcription by RNA polymerase II. Surprisingly, these foci coincide with the sites of polyA+RNA accumulation. Comparable colocalization and dependance on RNA polymerase II transcription is seen for the mtr1-1 mutant. The disorganization of the nucleolus thus depends on mRNA accumulation in these mutants. We discuss the possible functions of MTR2 and the yeast nucleolus in mRNA export.

Journal ArticleDOI
TL;DR: In transcriptionally active nuclei coiled bodies could serve as sites for initial preassembly and distribution of snRNP complexes for the three major RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre- mRNA 3' end formation.
Abstract: When demembranated sperm nuclei are placed in a Xenopus egg extract, they become surrounded by a nuclear envelope and then swell to form morphologically typical pronuclei. Granules ranging from < 1.0 to approximately 3.0 microns in diameter appear within such nuclei. Bell et al. identified four nucleolar proteins in these "prenucleolar bodies" by immunofluorescent staining (fibrillarin, nucleolin, B23/NO38, 180-kDa nucleolar protein). By in situ hybridization we show that these bodies also contain U3 and U8 small nuclear RNAs (snRNAs), known to be involved in pre-rRNA processing. Moreover, they contain all the snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6), as well as U7, which is required for histone pre-mRNA 3' end formation. In addition to the nucleolar antigens previously identified, we demonstrated staining with antibodies against the Sm epitope, trimethylguanosine, and coilin. Because the composition of these prenucleolar bodies is closer to that of coiled bodies than to nucleoli, we propose that they be referred to as coiled bodies. The existence of large coiled bodies in transcriptionally inactive pronuclei suggests that they may play a role in the import, assembly, and storage of RNA processing components but are not themselves sites of processing. In transcriptionally active nuclei coiled bodies could serve as sites for initial preassembly and distribution of snRNP complexes for the three major RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3' end formation.

Journal ArticleDOI
TL;DR: NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2 B NLS that is incompetent for nuclear localization in vivo, and it is shown that NPI46 is a nucleolar protein.
Abstract: We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae. The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro. Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo. NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Nopp140, and a COOH terminus with 45% homology to a family of mammalian and yeast proline isomerases. NPI46 is capable of catalyzing the prolyl cis-trans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay. By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein. NPI46 is not essential for cell viability.

Journal ArticleDOI
TL;DR: Two genes, encoding fibrillarin-like proteins from Methanococcus voltae and Methanitis vannielii, have been isolated and were named flpA (fibrillarian-like protein).
Abstract: Fibrillarin is found in the nucleolus of Eucarya and associated with small nucleolar RNAs. It is involved in the processing of precursor rRNA. Two genes, encoding fibrillarin-like proteins from Methanococcus voltae and Methanococcus vannielii, have been isolated. The genes were named flpA (fibrillarin-like protein).

Journal ArticleDOI
TL;DR: This study analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone, finding that despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.
Abstract: Programmed cell death is activated, by different stimuli and in many cell types, to regulate cell population balance during tissue proliferation and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca2+-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characterized by deep nuclear changes, consisting of typical cap-shaped chromatin marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late stage of the process and the greatest part of the phenomenon appears to take place despite good preservation of the plasma membrane and organellar component. In the present study we analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were processed for morphological examination in order to investigate the behaviour of the different nuclear domains. To follow DNA and RNA localization, we utilized osmium ammine and DNase-colloidal gold cytochemical reactions. The concentration of most DNA in the cap-shaped structures was demonstrated by these reactions. Confocal microscopy of cells processed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.

Journal ArticleDOI
TL;DR: A complex distribution of Rev in single cells was found and a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA is suggested.
Abstract: The human immunodeficiency virus type 1 (HIV-1) Rev (regulator of virion protein expression) protein exemplifies a new type of posttranscriptional regulation. One main function of Rev is to increase the cytoplasmic expression of unspliced and incompletely spliced retroviral mRNAs from which viral structural proteins are made. In that way, Rev is essential in order to complete the retroviral life cycle. The biology of Rev in the host cell has remained elusive. In this study, a complex distribution of Rev in single cells was found. Rev was found in the cytoplasm, in a perinuclear zone, in the nucleoplasm, and in the nucleoli. In the nucleoplasm, Rev colocalized in a speckled pattern with host cell factors known to assemble on nascent transcripts. Those factors are involved in the processing of heterogeneous RNA to spliced mRNA in the nucleoplasm of all cells. The distribution of Rev was dependent only on Rev and host cell interactions, since neither the Rev target RNA nor other HIV proteins were expressed in the cells. Rev was found in the same subcellular compartments of cells treated for extended periods with cycloheximide, an inhibitor of protein synthesis. This finding implies that Rev shuttles continuously between cytoplasmic and nucleoplasmic compartments. The results suggest a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA.

Journal ArticleDOI
TL;DR: These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations with the in situ activities of U3 and U13.
Abstract: The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.

Journal ArticleDOI
TL;DR: It is suggested that sphere organelles in the GV and coiled bodies in somatic nuclei are homologous organelle with similar functions in preassembly and sorting of RNA processing components, and differences in their composition suggest functional specialization in the two cell types.
Abstract: Cultured vertebrate cells often display one or more coiled bodies in their nuclei These are spherical structures approximately 05-10 micron in diameter that contain high concentrations of small nuclear ribonucleoproteins (snRNPs); they are distinct from nuclear speckles and nucleoli, the other major sites of snRNP concentration Coiled bodies in human cells contain a unique protein, p80-coilin, that has an M(r) = 80 kDa Cloned p80-coilin cDNA encodes 576 amino acids with a calculated molecular weight of 626 kDa To determine which of several snRNP-containing structures in the amphibian germinal vesicle (GV) might be the homologue of coiled bodies, we injected myc-tagged transcripts of full-length human p80-coilin into the cytoplasm of Xenopus oocytes and followed the fate of the translated proteins with an antibody specific for the tag Western blots of GV proteins showed rapid appearance of both full-length and truncated p80-coilin in the nucleus Immunofluorescent staining of spread GV contents demonstrated specific uptake of p80-coilin by the sphere organelle within 1 h after injection Similar experiments were performed with a series of deletion constructs that lacked progressively longer segments from the carboxy terminus A construct that contained only the first 102 amino acids (18% of the molecule) was specifically targeted to the sphere organelle Conversely, a construct lacking the first 92 amino acids failed to localize, although it was imported into the GV Thus, a relatively short region at the amino terminus of human p80-coilin is both necessary and sufficient for localization in the sphere organelle Sphere organelles in the GV and coiled bodies in somatic nuclei are clearly related in composition We suggest that they are homologous organelles with similar functions in preassembly and sorting of RNA processing components Differences in their composition suggest functional specialization in the two cell types

Journal ArticleDOI
TL;DR: The temporal change in An3 protein localization is consistent with a role in the production of large maternal pools of rRNA during oogenesis, similar to that of Xenopus oogenesis.
Abstract: An3 is a maternal mRNA localized to the animal hemisphere of oocytes and early embryos. We have analyzed the enzymatic activity and the subcellular localization of the protein encoded by An3 mRNA during Xenopus oogenesis. Antibodies raised using recombinant full-length and truncated An3 protein recognized a single protein in Xenopus and single proteins from HeLa cells, Drosophila, mouse testes, and Saccharomyces cerevisiae. An3 protein immunoprecipitated from stage IV and stage VI oocytes had ATP-dependent RNA helicase activity. The subcellular location of An3 protein changed during oocyte development. In previtellogenic oocytes, An3 was present throughout the nucleus; cytoplasmic localization was relatively sparse. Nuclear localization in mid-vitellogenic oocytes was primarily nucleolar; cytoplasmic staining increased relative to earlier stages. In stage VI oocytes, An3 protein was detected only in the cytoplasm. The temporal change in An3 protein localization is consistent with a role in the production of large maternal pools of rRNA during oogenesis.

Journal ArticleDOI
TL;DR: RNP complexes related to the processing steps of ribosome biogenesis in mammalian cells quit the nucleolus in late G2 and associate with the chromosome periphery until late telophase and both in the perichromosomal layer and the nucleolar remnant in CHO cells.

Journal ArticleDOI
TL;DR: Results show that expression of the intact human HSP72 or mutant human H SP72 missing its ATP-binding domain confers heat resistance and protects cells against heat-induced intranuclear protein aggregation.

Journal ArticleDOI
TL;DR: A new ultrastructural method for locating transcription on ultra-thin sections that has considerable advantages compared with current techniques, constituting a very useful tool to map transcriptionally active loci in a variety of cells.
Abstract: We describe a new ultrastructural method for locating transcription on ultra-thin sections. The use of anti-DNA/RNA hybrid antibodies provides specific labeling on precise structures of the nuclear...