Nucleolus

About: Nucleolus is a research topic. Over the lifetime, 5873 publications have been published within this topic receiving 232435 citations. The topic is also known as: GO:0005730 & cell nucleolus.

Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.

Abstract: The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

Nucleolar function and size in cancer cells.

Abstract: We have have studied the relationship between nucleolar function and size and cell doubling time in cancer cells. Seven human cancer cell lines characterized by different proliferation rates were used. Nucleolar functional activity was evaluated by measuring RNA polymerase I activity and expression of RNA polymerase I upstream binding factor (UBF), DNA topoisomerase I, and fibrillarin, three proteins involved in synthesis and processing of rRNA. Transcriptional activity of RNA polymerase I was strictly related to cell doubling time (r = -0.97; P < 0.001). The quantitative distribution of UBF, DNA topoisomerase I, and fibrillarin was evaluated on Western blots using specific monoclonal antibodies by densitometric analysis of autoradiographic signals. It was found to be directly related to RNA polymerase I transcriptional activity (r = 0.89, P = 0.008 for UBF; r = 0.95, P = 0.001 for DNA topoisomerase I; and r = 0.91, P = 0.004 for fibrillarin) and inversely related to cell doubling time (r = -0.87, P = 0.011 for UBF; r = -0.97, P < 0.001 for DNA topoisomerase I; and r = -0.91, P = 0.005 for fibrillarin). The nucleolar areas were measured by automated image analysis on toluidine blue-stained cells. The values of the stained nucleolar structures per cell were directly related to RNA polymerase I transcriptional activity (r = 0.94, P = 0.001) and inversely related to cell doubling time (r = -0.98, P < 0.001). The same area values of the nucleolar structures stained by toluidine blue were also closely related to the amount of UBF (r = 0.92, P = 0.003), DNA topoisomerase I (r = 0.98, P < 0.001), and fibrillarin (r = 0.95, P = 0.001), and to the in situ quantitative distribution of AgNOR proteins (r = 0.98, P < 0.001). Our results demonstrated that in cancer cells rRNA transcriptional activity and nucleolar size are inversely related to cell doubling time. Quantitative distribution of nucleolar structures within the cell represents a cytohistological parameter of the rapidity of cell proliferation.

Acrylamide gel electrophoresis of HeLa cell nucleolar RNA.

Abstract: The nucleolus of eucaryotic cells has been identified by Perry and others'-4 as the site of ribosomal RNA synthesis. It has recently become possible to obtain from HeLa cells a nucleolar preparation which, as seen by electron microscopy, is relatively free of chromatin.5' I Fractionation of C'4-uridine-labeled cells confirmed the hypothesis that the nucleolus is the site of synthesis of the 45S ribosomal RNA precursor.5 Another species of RNA (32S) is present in relatively large amounts. The nucleolus appears to contain only ribosomal precursor RNA since if fractionation is performed carefully, very little of the nucleoplasmic heterodisperse RNA is associated with it.7 8 The following picture of the major events in ribosomal RNA formation has emerged. The initial event is the synthesis of a high-molecularweight precursor molecule with a sedimentation constant of 45S.Y-1 After 15 to 20 minutes, this molecule is cleaved, yielding 18S ribosomal RNA and a species of RNA whose sedimentation constant is 32S.12 The 18S RNA is quickly transported from the nucleolus and appears in the cytoplasm as part of the smaller ribosomal subunit. After additional processing time in the nucleolus, the 32S molecule is converted to 28S and eventually emerges into the cytoplasm as part of the larger ribosomal subunit. With the development by Loening of a method of polyacrylamide gel electrophoresis for molecules as large as ribosomal RNA, 3 it has become possible to study in greater detail the events of nucleolar RNA processing. The gels used in these experiments have been modified by the addition of glycerol to facilitate freezing and slicing. The following information has been obtained concerning nucleolar processing of ribosomal RNA. (1) The site of transformation of 32S to 28S RNA is the nucleolus. (2) Several additional short-lived intermediate species of ribosomal RNA have been identified with estimated sedimentation constants of 41S, 36S, and 20S. (3) Some short-lived intermediates increase in amount under conditions that disrupt normal nucleolar RNA processing, e.g., poliovirus infection. (4) The conversion of 45S RNA to mature ribosomal RNA is accompanied by a net loss of RNA. This is also shown for the transformation of 32S RNA to 28S. Materials and Methods.-Cells: HeLa type 3 cells were grown and labeled in suspension culture as previously described.14 Radioisotopes: Imethionine-methyl-C'4 (49 mc/mM) and uridine-2-C'4 (27 mc/mM) were purchased from Schwarz BioResearch. Imethionine-methyl-H3 (1400 me/mM) was purchased from Nuclear Chicago. Methyl labeling was performed in Eagle's medium free of unlabeled methionine and containing adenosine and guanosine (2 X 10-5 M). When noted, unlabeled methionine was added back to this medium to prevent methionine starvation. Cell fractionation: Cells were separated into nuclear and cytoplasmic fractions as previously described.1' The cleaned nuclei from approximately 4 X 107 cells were suspended in 1 ml of highionic-strength buffer (HSB: 0.5 M NaCl, 0.05 M MgCl2, 0.01 M Tris, pH 7.4), warmed briefly to 370, and digested with 50 yg of Worthington electrophoretically purified DNase. The digest

Dynamics of human DNA topoisomerases IIα and IIβ in living cells

Abstract: DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (α and β) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIα and IIβ behaved similarly in interphase but differently in mitosis, where only topo IIα was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIα was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.

Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity

Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are the two common neurodegenerative diseases that have been associated with the GGGGCC·GGCCCC repeat RNA expansion in a noncoding region of C9orf72. It has been previously reported that unconventional repeat-associated non-ATG (RAN) translation of GGGGCC·GGCCCC repeats produces five types of dipeptide-repeat proteins (referred to as RAN proteins): poly-glycine-alanine (GA), poly-glycine-proline (GP), poly-glycine-arginine (GR), poly-proline-arginine (PR) and poly-proline-alanine (PA). Although protein aggregates of RAN proteins have been found in patients, it is unclear whether RAN protein aggregation induces neurotoxicity. In the present study, we aimed to understand the biological properties of all five types of RAN proteins. Surprisingly, our results showed that none of these RAN proteins was aggregate-prone in our cellular model and that the turnover of these RAN proteins was not affected by the ubiquitin-proteasome system or autophagy. Moreover, poly-GR and poly-PR, but not poly-GA, poly-GP or poly-PA, localized to the nucleolus and induced the translocation of the key nucleolar component nucleophosmin, leading to nucleolar stress and cell death. This poly-GR- and poly-PR-mediated defect in nucleolar function was associated with the suppression of ribosomal RNA synthesis and the impairment of stress granule formation. Taken together, the results of the present study suggest a simple model of the molecular mechanisms underlying RAN translation-mediated cytotoxicity in C9orf72-linked ALS/FTD in which nucleolar stress, but not protein aggregation, is the primary contributor to C9orf72-linked neurodegeneration.