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The nucleolus: structure/function relationship in RNA metabolism

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Abstract The nucleolus is the ribosome factory of the cells. This is the nuclear domain where ribosomal RNAs are synthesized, processed, and assembled with ribosomal proteins. Here we describe the classical tripartite organization of the nucleolus in mammals, reflecting ribosomal gene transcription and pre‐ribosomal RNA (pre‐rRNA) processing efficiency: fibrillar center, dense fibrillar component, and granular component. We review the nucleolar organization across evolution from the bipartite organization in yeast to the tripartite organization in humans. We discuss the basic principles of nucleolar assembly and nucleolar structure/function relationship in RNA metabolism. The control of nucleolar assembly is presented as well as the role of pre‐existing machineries and pre‐rRNAs inherited from the previous cell cycle. In addition, nucleoli carry many essential extra ribosomal functions and are closely linked to cellular homeostasis and human health. The last part of this review presents recent advances in nucleolar dysfunctions in human pathology such as cancer and virus infections that modify the nucleolar organization. Copyright © 2010 John Wiley & Sons, Ltd. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry Translation > Ribosome Biogenesis RNA in Disease and Development > RNA in Disease

Nucleolar organization of a human HeLa cell prepared by conventional methods for electron microscopy. The cells were fixed by glutaraldehyde and osmic acid. The sections were contrasted by uranyl acetate and lead citrate. (a) Section of one nucleolus and (b) details of the three nucleolar components. In (a) and (b), the three nucleolar components are visible: the fibrillar centers (asterisks), the dense fibrillar component (white arrow), and granular component (GC). Scale bar: (a) = 0.5 µm and (b) = 0.1 µm.

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In cycling cells, nucleolar assembly takes about 2 h. In HeLa cells, transcription by pol I starts in telophase in the six active NORs, whereas the mitotic chromatin is still condensed (illustrated as two oval dark structure). In early G1a, the mitotic chromatin decondenses (illustrated in grey), the nuclear envelope (broken line) is assembled, numerous PNBs (dark foci) are formed, and the active NORs recruit the processing proteins in DFC (green). In early G1b, the processing proteins are almost completely transferred from PNBs to GC, and NORs regrouped in two to three nucleoli. Interphasic cells are generated, when the cytoplasmic bridge (not shown) between the two daughter cells is broken. GC, granular component; NOR, nucleolar organizer regions; PNBs, prenucleolar bodies.

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Nucleolar organization at the transition between bipartite and tripartite organization. (a) A nucleolus from Trachemys scripta (red‐eared slider) and (b) a nucleolus from Podarcis muralis (common wall lizard). All samples treated by acetylation and inspected by EM. Scale bars = 0.4 µm. EM, electron microscopy.

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Nucleolar organization across evolution. Lower and higher eukaryotes are characterized by a bipartite (F and G) versus a tripartite (FC, DFC, and GC) nucleolar organization, respectively, as illustrated under physiological (a and c) and segregation (b and d) conditions. (a) A wild‐type yeast nucleolus with fibrillar strands (F) and granules (G). (b) A yeast nucleolus from a cell deleted for srp40, the two nucleolar components are segregated and adopt a ‘Ying‐Yang’ configuration. (c) A wild‐type human nucleolus with several FC/DFC (asterisks and arrows) modules embedded into a single GC. (d) A human nucleolus following actinomycin D treatment (0.5 µg/mL, 2 h) with all three components segregated. All samples were treated by acetylation and inspected by EM. (a, b) Yeast Saccharomyces cerevisiae; (c) HEp‐2 larynx carcinoma; and (d) Jurkat T lymphocyte. Scale bars = 0.2 µm.

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Organization of the nucleolus after inhibition of pol I transcription. In light microscopy, the nucleolar segregation in a human HeLa cell treated with a low concentration of actinomycin D is observed in the left panel. (a) The nucleolus is visible in the nucleus by phase contrast. (b–d) The DFC visualized by fibrillarin–GFP fusion and the GC visualized by NPM/B23‐DsRed fusion disengage and form two juxtaposed structures. In the right panel, (e) the segregation of the three nucleolar components observed in electron microscopy. This HeLa cell was treated with low concentration of actinomycin D and the Ag‐NOR staining (black dots) revealed the Ag‐NOR proteins in FC. DFC, dense fibrillar component; FC, fibrillar center; GC, granular component. Scale bar = 1 µm.

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Localization of nucleolar markers in the three nucleolar components in human HeLa cells. In the right panel, the green labelings show the distribution of the proteins and in the left panel in the same cells the nucleoli are visible in dark by phase contrast. Antibodies against UBF decorate several foci in the nucleolar interior corresponding to FCs. Nopp140‐GFP (Nopp140) fusions exhibit a dotted labeling characteristic of the DFC of nucleoli. NPM/B23‐GFP fusions (NPM/B23) decorate the GC. Scale bar: 5 µm. DFC, dense fibrillar component; FCs, fibrillar centers; GC, granular component; UBF, upstream binding factor.

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GFC in neuron nucleolus. (a) The confocal image illustrates a trigeminal ganglia neuron immunostained for UBF (green) and counterstained with propidium iodide for nucleic acids. Two cells of different size are visible: the large cell corresponds to the neuron and the asterisk indicates the nucleus of a satellite glial cell. The nucleolus (arrow head) of the large neuron contains a prominent GFC visible in green and several FCs of normal size. In the enlargement (left corner) of the nucleolus, the FCs of normal size are indicated by arrows and the GFC is visible in the center. Scale bar = 5 µm. (b) Nucleolus in a trigeminal ganglia neuron observed in EM. A typical GFC is visible in the center of the nucleolus. Scale bar = 1 µm. Unpublished data from the group of M. Lafarga (I. Casafont and M. T. Berciano). EM, electron microscopy; FC, fibrillar center; GFC, giant FC; UBF, upstream binding factor.

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Perinucleolar heterochromatin in mouse NIH3T3 nuclei observed in light microscopy and EM are shown in left and right panels, respectively. (a–c) The heterochromatin is observed after DNA Dapi staining especially at the nucleolar periphery (arrows); the nucleoli (contrasted structure in phase) appeared as black holes with Dapi. (d) A protocol to preferentially reveal the nucleic acids in EM was used. The DNAs and RNAs were contrasted with uranyl after methylation and acetylation of the amino and carboxyl groups. Around the nucleolus, two large clumps of chromatin (arrow heads) are visible as well as the perinucleolar chromatin. White arrows indicate intranucleolar chromatin localized in the GC. One FC is visible in the middle of the nucleolus. Scale bar = 0.5 µm. EM, electron microscopy; FC, fibrillar center.

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