Catching RNA Polymerase I in Flagranti: Ribosomal Genes Are Transcribed in the Dense Fibrillar Component of the Nucleolus

1995 ◽  
Vol 216 (2) ◽  
pp. 285-289 ◽  
Author(s):  
Pavel Hozák
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Genes ◽  
Rna Polymerase I ◽  
Dense Fibrillar Component ◽  
Fibrillar Component ◽  
Polymerase I

scholarly journals Localisation of the Ki-67 antigen within the nucleolus. Evidence for a fibrillarin-deficient region of the dense fibrillar component

1996 ◽  
Vol 109 (6) ◽  
pp. 1253-1263 ◽  
Author(s):  
I.R. Kill
Keyword(s):  
Rna Polymerase ◽  
Indirect Immunofluorescence ◽  
Dermal Fibroblasts ◽  
Rna Polymerase I ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dense Fibrillar Component ◽  
Ki 67 ◽  
Fibrillar Component ◽  
Polymerase I

The Ki-67 antigen is detected in proliferating cells in all phases of the cell division cycle. Throughout most of interphase, the Ki-67 antigen is localised within the nucleous. To learn more about the relationship between the Ki-67 antigen and the nucleolus, we have compared the distribution of Ki-67 antibodies with that of a panel of antibodies reacting with nucleolar components by confocal laser scanning microscopy of normal human dermal fibroblasts in interphase stained in a double indirect immunofluorescence assay. During early G1, the Ki-67 antigen is detected at a large number of discrete foci throughout the nucleoplasm, extending to the nuclear envelope. During S-phase and G2, the antigen is located in the nucleolus. Double indirect immunofluorescence studies have revealed that during early to mid G1 the Ki-67 antigen is associated with reforming nucleoli within discrete domains which are distinct from domains containing two of the major nucleolar antigens fibrillarin and RNA polymerase I. Within mature nucleoli the Ki-67 antigen is absent from regions containing RNA polymerase I and displays only partial co-localisation within domains containing either fibrillarin or B23/nucleophosmin. Following disruption of nucleolar structure, induced by treatment of cells with the drug 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or with actinomycin D, the Ki-67 antigen translocates to nucleoplasmic foci which are associated with neither fibrillarin nor RNA polymerase I. However, in treated cells the Ki-67 Ag remains associated with, but not co-localised to, regions containing B23/nucleophosmin. Our observations suggest that the Ki-67 antigen associates with a fibrillarin-deficient region of the dense fibrillar component of the nucleolus. Integrity of this region is lost following either nucleolar dispersal or nucleolar segregation.

scholarly journals Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells.

1987 ◽  
Vol 105 (4) ◽  
pp. 1483-1491 ◽  
Author(s):  
R Benavente ◽  
K M Rose ◽  
G Reimer ◽  
B Hügle-Dörr ◽  
U Scheer
Keyword(s):  
Rna Polymerase ◽  
Cultured Cells ◽  
Active Form ◽  
Nucleolar Organizer ◽  
Rna Polymerase I ◽  
Nucleolar Organizer Regions ◽  
Dense Fibrillar Component ◽  
Nucleolar Material ◽  
Fibrillar Component ◽  
Polymerase I

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.

A Drosophila anti-RNA polymerase II antibody recognizes a plant nucleolar antigen, RNA polymerase I, which is mostly localized in fibrillar centres

1991 ◽  
Vol 100 (1) ◽  
pp. 99-107 ◽  
Author(s):  
M. Martin ◽  
F.J. Medina
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Dense Fibrillar Component ◽  
Transition Area ◽  
Fibrillar Component ◽  
Polymerase I

The distribution of nucleolar RNA polymerase in the nucleolus of onion root meristematic cells has been studied by means of an antibody originally raised against Drosophila RNA polymerase II. This antibody recognizes the homologous domains of the large subunit of the enzyme, which are highly conserved throughout evolution in the three classes of eucaryotic RNA polymerases. Given that RNA polymerase I is confined to the nucleolus, and that the onion cell nucleolus lacks digitations of extranucleolar chromatin, we conclude that the nucleolar enzyme localized is RNA polymerase I. A quantitative approach, independent of the existence of borderlines between nucleolar fibrillar centres and the dense fibrillar component, allowed us to show that the enzyme is localized in fibrillar centres and in the transition area between them and the dense fibrillar component, in parallel with the nucleolar DNA. These results, together with previous autoradiographic, cytochemical and immunocytochemical results, in this and other species, lead us to conclude that the activation of rDNA for transcription occurs in the fibrillar centres and pre-rRNA synthesis is expressed at the transition area between fibrillar centres and the dense fibrillar component. Fibrillar centres are connected to each other by extended RNA polymerase-bound DNA fibres, presumably active in transcription. This work provides evidence of the high evolutionary conservation of some domains of the large subunit of RNA polymerases and of the existence of fibrillar centres in the nucleolus of plant cells, totally homologous to those described in mammalian cells.

Transcription of yeast DNA by homologous RNA polymerases I and II: selective transcription of ribosomal genes by RNA polymerase I

Biochemistry ◽  
1977 ◽  
Vol 16 (1) ◽  
pp. 16-24 ◽  
Author(s):  
Michael J. Holland ◽  
Gordon L. Hager ◽  
William J. Rutter
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Genes ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Polymerase I

scholarly journals Assembly and Functional Organization of the Nucleolus: Ultrastructural Analysis ofSaccharomyces cerevisiaeMutants

2000 ◽  
Vol 11 (6) ◽  
pp. 2175-2189 ◽  
Author(s):  
Stéphanie Trumtel ◽  
Isabelle Léger-Silvestre ◽  
Pierre-Emmanuel Gleizes ◽  
Frédéric Teulières ◽  
Nicole Gas
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Functional Organization ◽  
Ultrastructural Localization ◽  
Rna Polymerase I ◽  
Wild Type ◽  
Rdna Transcription ◽  
Nucleolar Structure ◽  
Fibrillar Component ◽  
Polymerase I

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.

lncRNA SLERT controls phase separation of FC/DFCs to facilitate Pol I transcription

Science ◽  
2021 ◽  
Vol 373 (6554) ◽  
pp. 547-555
Author(s):  
Man Wu ◽  
Guang Xu ◽  
Chong Han ◽  
Peng-Fei Luan ◽  
Yu-Hang Xing ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Noncoding Rna ◽  
Rna Polymerase I ◽  
Dense Fibrillar Component ◽  
Biophysical Properties ◽  
Fibrillar Center ◽  
Dead Box ◽  
Pol I ◽  
Fibrillar Component ◽  
Polymerase I

RNA polymerase I (Pol I) transcription takes place at the border of the fibrillar center (FC) and the dense fibrillar component (DFC) in the nucleolus. Here, we report that individual spherical FC/DFC units are coated by the DEAD-box RNA helicase DDX21 in human cells. The long noncoding RNA (lncRNA) SLERT binds to DDX21 RecA domains to promote DDX21 to adopt a closed conformation at a substoichiometric ratio through a molecular chaperone–like mechanism resulting in the formation of hypomultimerized and loose DDX21 clusters that coat DFCs, which is required for proper FC/DFC liquidity and Pol I processivity. Our results suggest that SLERT is an RNA regulator that controls the biophysical properties of FC/DFCs and thus ribosomal RNA production.

Psoralen photocrosslinking, a tool to study the chromatin structure of RNA polymerase I - transcribed ribosomal genes

2005 ◽  
Vol 83 (4) ◽  
pp. 449-459 ◽  
Author(s):  
Martin Toussaint ◽  
Geneviève Levasseur ◽  
Maxime Tremblay ◽  
Michel Paquette ◽  
Antonio Conconi
Keyword(s):  
Rna Polymerase ◽  
Chromatin Structure ◽  
Nuclear Dna ◽  
Rdna Locus ◽  
Ribosomal Genes ◽  
Rna Polymerase I ◽  
Coding Regions ◽  
Polymerase I

The chromatin structure of RNA polymerase I - transcribed ribosomal DNA (rDNA) is well characterized. In most organisms, i.e., lower eukaryotes, plants, and animals, only a fraction of ribosomal genes are transcriptionally active. At the chromatin level inactive rDNA is assembled into arrays of nucleosomes, whereas transcriptionally active rDNA does not contain canonical nucleosomes. To separate inactive (nucleosomal) and active (non-nucleosomal) rDNA, the technique of psoralen photocrosslinking has been used successfully both in vitro and in vivo. In Saccharomyces cerevisiae, the structure of rDNA chromatin has been particularly well studied during transcription and during DNA replication. Thus, the yeast rDNA locus has become a good model system to study the interplay of all nuclear DNA processes and chromatin. In this review we focused on the studies of chromatin in ribosomal genes and how these results have helped to address the fundamental question: What is the structure of chromatin in the coding regions of genes?Key words: active chromatin, FACT, lexosome, psoralen, photo-crosslinking, rDNA, RNA polymerase I.

Three-dimensional organization of active rRNA genes within the nucleolus

2002 ◽  
Vol 115 (16) ◽  
pp. 3297-3307 ◽  
Author(s):  
Thierry Cheutin ◽  
Marie-Françoise O'Donohue ◽  
Adrien Beorchia ◽  
Marc Vandelaer ◽  
Hervé Kaplan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Ultrastructural Level ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Fibrillar Component ◽  
Polymerase I

In this work, we have localized transcribing rRNA genes at the ultrastructural level and described their three-dimensional organization within the nucleolus by electron tomography. Isolated nucleoli, which exhibit a reduced transcriptional rate, were used to determine the sites of initial BrUTP incorporation (i.e. rRNA synthesis by the transcriptional machinery). Using pulse-chase experiments with BrUTP and an elongation inhibitor,cordycepin, it was possible to precisely localize the initial sites of BrUTP incorporation. Our data show that BrUTP incorporation initially takes place in the fibrillar centers and that elongating rRNAs rapidly enter the surrounding dense fibrillar component. Furthermore, we investigated the spatial arrangement of RNA polymerase I molecules within the whole volume of the fibrillar centers. Electron tomography was performed on thick sections of cells that had been labeled with anti-RNA polymerase I antibodies prior to embedding. Detailed tomographic analyses revealed that RNA polymerase I molecules are mainly localized within discrete clusters. In each of them, RNA polymerase I molecules were grouped as several coils, 60 nm in diameter. Overall, these findings have allowed us to propose a model for the three-dimensional organization of transcribing rDNA genes within the nucleolus.

Localization of the RNA polymerase I transcription factor hUBF during the cell cycle

1993 ◽  
Vol 104 (2) ◽  
pp. 327-337 ◽  
Author(s):  
P. Roussel ◽  
C. Andre ◽  
C. Masson ◽  
G. Geraud ◽  
D. Hernandez-Verdun
Keyword(s):  
Rna Polymerase ◽  
Nucleolar Organizer ◽  
Ribosomal Gene ◽  
Human Cells ◽  
Rna Polymerase I ◽  
Nucleolar Organizer Regions ◽  
3 Dimensional ◽  
Definitive Evidence ◽  
Fibrillar Component ◽  
Polymerase I

Autoantibodies directed against nucleoli that recognized a doublet of 97–94 kDa in HeLa nuclear protein extracts were identified. The two polypeptides bound equal amounts of antibody, and each was recognized by antibodies affinity purified using the other polypeptide. These antigens were localized in the secondary constriction of PtK1 cells, i.e. the nucleolar organizer regions (NORs) where ribosomal genes accumulate. They were observed in human cells in the same sites as the NOR-silver-stained proteins. The molecular mass of the antigens, their characteristics in Western blotting and their localization in nucleoli and NORs during mitosis are consistent with them being RNA polymerase I transcriptional factor, UBF. This identification was confirmed on Western blotted proteins by their identical labelling patterns, using these autoantibodies and an anti-mUBF antibody that had been previously described. We obtained definitive evidence that these autoantibodies recognize UBF by the strong positive labelling of purified hUBF (1 to 4 ng). During interphase, these autoantibodies directed against UBF labelled in a folded filament pattern as small beads that may correspond to individual transcriptional units. In electron microscopy, the antibodies were observed in the dense fibrillar component (DFC) of the nucleoli and at the periphery of the fibrillar centers (FCs). At the end of G2 phase, transcription inactivation was concomitant with the gathering of UBF at mitotic NORs. UBF was not equally distributed between NORs in human cells: some NORs scored negative (2 to 4) and the intensity of labelling of positive NORs (6 to 8) differed. In confocal microscopy, 3-dimensional analysis of mitosis indicated that UBF remained associated with NORs during all mitotic stages and that there was equal partition of UBF between the daughter cells. The relationship between proteins associated with the NORs and ribosomal gene transcription is discussed.

The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes

Chromosoma ◽  
1993 ◽  
Vol 102 (9) ◽  
pp. 599-611 ◽  
Author(s):  
Olga V. Zatsepina ◽  
Renate Voit ◽  
Ingrid Grummt ◽  
Herbert Spring ◽  
Michael V. Semenov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Ribosomal Genes ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Transcription Initiation Factor ◽  
Polymerase I
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