Relative distribution of rDNA and proteins of the RNA polymerase I transcription machinery at chromosomal NORs

Suja, José Á.; Gébrane-Younès, Jeannine; Géraud, Gérard; Hernandez‐Verdun, Danièle

doi:10.1007/s004120050208

Cited by 7 publications

(8 citation statements)

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“…Interestingly, this domain plays a similar role at mitosis, where it associates topoisomerase I with the nucleolar organizing regions of mitotic chromosomes, a finding previously also indicated by immunohistochemistry (28,29). At both locations (fibrillar centers of the nucleolus and nucleolar organizing regions) topoisomerase I co-localizes with RNA polymerase I, which is believed to form a holo-enzyme together with a variety of other proteins also involved in rRNA transcription (30).…”

Section: Discussionmentioning

confidence: 61%

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

Christensen

Barthelmes

Boege

et al. 2002

Journal of Biological Chemistry

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DNA topoisomerase I releases torsion stress created by DNA transcription. In principle, this activity is required in the nucleoplasm for mRNA synthesis and in the nucleoli for rRNA synthesis. Yet, topoisomerase I is mostly a nucleolar protein. Current belief holds that this preference is triggered by the N-terminal domain of the enzyme, which constitutes a nucleolar import signal. Contradicting this view, we show here that nucleolar accumulation of various fragments of topoisomerase I is correlated with their lesser mobility in this compartment and not with the N-terminal domain being intact or present. Therefore, the N-terminal domain is not likely a nucleolar import signal. We show that it rather serves as an adaptor that anchors a subpopulation of topoisomerase I at fibrillar centers of nucleoli and nucleolar organizer regions of mitotic chromosomes. Thus, it provides a steady association of topoisomerase I with the rDNA and with RNA polymerase I, which is maintained in a living cell during the entire cell cycle.DNA topoisomerase I changes the pitch of DNA helices by cutting one DNA strand and allowing rotation of the other (1). One important role for this mechanism is the release of torsion stress created by DNA transcription. Thus, topoisomerase I activity should in principle be required in the nucleoplasm for the synthesis of mRNA, and in the nucleolus for the synthesis of rRNA. However, the latter task seems to dominate. During interphase, most of topoisomerase I is located in the nucleoli and not in the nucleoplasm, although the enzyme interchanges constantly between these two compartments (2). It has been proposed that nucleolar accumulation of topoisomerase I reflects engagement in rDNA-transcription because the rRNA genes are by far the most heavily transcribed genes in the cell. In keeping with this, studies of genomic DNA cleavage by topoisomerase I have invariantly come up with sites in the rDNA (3-5). However, catalytic interactions with rDNA cannot per se account for the accumulation of topoisomerase I in the nucleoli because stabilization of covalent topoisomerase I⅐DNA intermediates by camptothecin immobilizes the enzyme preferentially at nucleoplasmatic sites and only to a much lesser extent in the nucleoli (2). Along this reasoning the question arises of what restricts topoisomerase I in the nucleolus and how is this phenomenon related to rDNA processing. We show here that in a living cell a sub population of topoisomerase I is constantly anchored at the fibrillar centers of the nucleolus or the nucleolar organizer region of mitotic chromosomes and that this is due to an adaptor function of the N-terminal domain of the enzyme. EXPERIMENTAL PROCEDURESCloning and Cell Culture-GFP 1 chimera of human topoisomerase I and of various fragments of it (see Fig. 3A) were stably expressed in the human embryonal kidney cell line 293 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) using the bicistronic expression vector pMC-2P (6) in which the translational initiation of the...

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Section: Discussionmentioning

confidence: 61%

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

Christensen

Barthelmes

Boege

et al. 2002

Journal of Biological Chemistry

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“…Thus, topo I does not make an exception to the general rule that molecules are targeted to the nucleolus due to their interaction with one of the three nucleolar building blocks that are structured around rDNA and/or its transcripts (35)(36)(37). Even during mitosis, where rDNA is not transcribed, topo I remains associated with the corresponding chromosomal structures that harbor rDNA and RNA polymerase I (5,38,39). Thus, positioning of topo I is crucially determined by its co-localization with rDNA and RNA polymerase I, and this co-localization is maintained throughout the entire cell cycle irrespectively of where in the nucleus these complexes are localized and independently of ongoing rRNA transcription or active DNA turnover by topo I itself.…”

Section: Discussionmentioning

confidence: 99%

Distinct Effects of Topoisomerase I and RNA Polymerase I Inhibitors Suggest a Dual Mechanism of Nucleolar/Nucleoplasmic Partitioning of Topoisomerase I

Christensen

Krokowski

Barthelmes

et al. 2004

Journal of Biological Chemistry

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Topoisomerase I is mostly nucleolar, because it plays a preeminent role in ribosomal DNA (rDNA) transcription. It is cleared from nucleoli following exposure to drugs stabilizing covalent DNA intermediates of the enzyme (e.g. camptothecin) or inhibiting RNA polymerases (e.g. actinomycin D), an effect summarily attributed to blockade of rDNA transcription. Here we show that two distinct mechanisms are at work: (i) Both drugs induce inactivation and segregation of the rRNA transcription machinery. With actinomycin D this leads to a co-migration of RNA-polymerase I and topoisomerase I to the nucleolar perimeter. The process has a slow onset (>20 min), is independent of topoisomerase I activity, but requires the N-terminal domain of the enzyme to colocalize with RNA polymerase I. (ii) Camptothecin induces, in addition, immobilization of active topoisomerase I on genomic DNA resulting in rapid nucleolar clearance and spreading of the enzyme to the entire nucleoplasm. This effect is independent of the state of rRNA transcription, involves segregation of topoisomerase I from RNA polymerase I, has a rapid onset (<1 min), and requires catalytic activity but neither the N-terminal domain of topoisomerase I nor its major sumoylation site. Thus, nucleolar/nucleoplasmic partitioning of topoisomerase I is regulated by interactions with RNA polymerase I and DNA but not by sumoylation.The mechanism that distributes DNA-topoisomerase I (topo I) 1 between nucleoli and nucleoplasm has been a controversial issue over a decade. Topoisomerase I catalyzes topological changes in the DNA by cutting one strand of the double helix and allowing rotation of the other (1). This mechanism releases torsion stress in DNA double helices that is created by e.g. RNA synthesis. Thus, topo I is a crucial cofactor for the transcription of nucleoplasmic genes (2) and rDNA (3, 4). The latter task seems to dominate, because topo I is concentrated in the nucleoli, and, of the three nucleolar components, it is preferentially found in the fibrillar centers, a restriction in localization that is lost when the N-terminal domain of the enzyme or parts of it are lacking (5). Topoisomerase I is thus placed in the vicinity of rDNA, RNA polymerase I, and rDNA transcription, which are also restricted to the fibrillar centers of the nucleolus (6). Nucleolar positioning of topo I is highly susceptible to exogenous perturbation. The enzyme is lost from fibrillar centers, when cells are cultured at an inappropriate temperature (5). It is also lost from nucleoli in response to camptothecin and related compounds that inhibit the religation step of the DNA cleavage-religation mechanism and thus stabilize the enzyme in covalent DNA intermediates (7,8). Because such compounds also inhibit RNA synthesis (9) and because direct inhibitors of RNA polymerase I such as actinomycin D (10) also remove topo I from the nucleoli, it has been proposed that the same cellular mechanism (i.e. loss of rRNA synthesis) is responsible for nucleolar clearance of topo I in response to both typ...

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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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Relative distribution of rDNA and proteins of the RNA polymerase I transcription machinery at chromosomal NORs

Cited by 7 publications

References 0 publications

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

Distinct Effects of Topoisomerase I and RNA Polymerase I Inhibitors Suggest a Dual Mechanism of Nucleolar/Nucleoplasmic Partitioning of Topoisomerase I

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

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