Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Héliot, Laurent; Kaplan, Hervé; Lucas, Laurent; Klein, Christophe; Beorchia, A.; Doco‐Fenzy, Martine; Ménager, M.; Thiry, Marc; O’Donohue, Marie-Françoise; Ploton, Dominique

doi:10.1091/mbc.8.11.2199

Cited by 72 publications

(60 citation statements)

References 49 publications

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“…On the one hand, essential components of the rRNA transcription machinery, including the upstream binding factor (UBF), the subunits of promoter selectivity factor SL1, as well as RNA pol I, are present at the NORs of chromosomes (Weisenberger and Scheer, 1995;Jordan et al, 1996;Roussel et al, 1996;Seither et al, 1997). In addition, UBF, RNA pol I, and DNA topoisomerase I were found in the vicinity of previously active rDNA genes at NORs during metaphase and anaphase (Gé-brane-Younès et al, 1997;Heliot et al, 1997;Suja et al, 1997). On the other hand, nascent pre-rRNA molecules have not been detected in the chromosomal NORs.…”

Section: Discussionmentioning

confidence: 99%

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Dundr

Olson

1998

MBoC

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Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5Ј-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5Ј-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3Ј-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli. INTRODUCTIONThe nucleolus is a prominent membraneless subnuclear compartment assembled around clusters of tandemly repeated rDNA genes from which prerRNA is transcribed and subsequently folded, processed, modified, and assembled into small and large ribosomal subunits (Scheer et al., 1993;Shaw and Jordan, 1995;Maden and Hughes, 1997). A remarkable aspect of the cell cycle is the disintegration of the nucleolus during mitosis and its reassembly as the daughter cells proceed toward G 1 phase. A pivotal event in this process is the repression of RNA polymerase (pol) I-driven pre-rRNA synthesis between prophase and telophase (Prescott and Bender, 1962). At the same time, nucleolar components disperse to various subcellular locations as the maternal nucleoli diassemble (Goessens, 1984).A comprehensive understanding of the locations and fates of nucleolar components during mitosis is slowly emerging (Scheer et al., 1993;Hernandez-Verdun and Gautier, 1994). For example, much of the RNA pol I transcription machinery and DNA topoisomerase I remain associated with the nucleolus organizer regions (NORs) 1 of chromosomes between nucleolar disassembly in late prophase and reassembly in telophase (Scheer et al., 1993; Weisenberger and * Corresponding author. 1 Abbrevations used: DFC, dense fibrillar component; ETS, external transcribed spacer; GC, granular component; ITS, internal transcribed spacer; NDF, nucleolus-derived foci; NOR, nucleolus organizer region; PNB, prenucleolar body; pol, polymerase; snoRNA, small nucleolar RNA; sno-RNP, small nucleolar ribonucleoprotein.© 1998 by The American Society for Ce...

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Section: Discussionmentioning

confidence: 99%

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Dundr

Olson

1998

MBoC

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“…(Derenzini et al 1983c;Hernandez-Verdun et al 1984;Puvion-Dutilleul et al 1992). Moreover, when RNA synthesis is stopped during mitosis, the DNA present in the metaphase NORs is always maintained in an extended non-nucleosomal configuration (HernandezVerdun and Derenzini 1983;Heliot et al 1997).…”

Section: Structural-functional Relationship Of Ribosomal Genes In Situmentioning

confidence: 99%

Structural and Functional Organization of Ribosomal Genes within the Mammalian Cell Nucleolus

Derenzini

Pasquinelli

O’Donohue

et al. 2006

J Histochem Cytochem.

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S U M M A R Y Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA doublehelix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell. (J Histochem Cytochem 54:131-145, 2006)

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“…During metaphase, NORs that were transcriptionally active in the previous interphase form prominent chromosomal features termed secondary constrictions, which appear as achromatic gaps when stained with Giemsa or AT-specific fluorochromes such as DAPI (4Ј,6-diamidino-2-phenylindole) or quinacrine (Sumner 1982). Electron tomography revealed that chromatin within secondary constrictions is 10-fold less condensed than the rest of the chromosome (Heliot et al 1997). Classically, secondary constrictions are positive in silver staining and often termed AgNORs (Goodpasture and Bloom 1975).…”

mentioning

confidence: 99%

UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery

Mais¹,

Wright²,

Prieto³

et al. 2004

Genes Dev.

164

191

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Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein-protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements.[Keywords: RNA polymerase I; ribosomal DNA; nucleolar organiser region; nucleolus; upstream-binding factor] Supplemental material is available at http://www.genesdev.org.

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Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Cited by 72 publications

References 49 publications

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Structural and Functional Organization of Ribosomal Genes within the Mammalian Cell Nucleolus

UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery

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