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.

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 role for upstream binding factor in organizing ribosomal gene chromatin

2006 ◽  
Vol 73 ◽  
pp. 77-84 ◽  
Author(s):  
Jane E. Wright ◽  
Christine Mais ◽  
José-Luis Prieto ◽  
Brian McStay
Keyword(s):  
Nucleolar Organizer ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Nucleolar Organizer Regions ◽  
Binding Factor ◽  
Upstream Binding Factor ◽  
Acrocentric Chromosomes ◽  
Polymerase I ◽  
Secondary Constrictions ◽  
Active Nors

Human ribosomal genes are located in NORs (nucleolar organizer regions) on the short arms of acrocentric chromosomes. During metaphase, previously active NORs appear as prominent chromosomal features termed secondary constrictions, which are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I transcription factor UBF (upstream binding factor) binds extensively across the ribosomal gene repeat throughout the cell cycle. Evidence that UBF underpins NOR structure is provided by an examination of cell lines in which large arrays of a heterologous UBF binding sequences are integrated 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, that are morphologically similar to NORs.

scholarly journals UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity

Nucleus ◽  
2013 ◽  
Vol 4 (6) ◽  
pp. 478-486 ◽  
Author(s):  
Margarita Sobol ◽  
Sukriye Yildirim ◽  
Vlada V Philimonenko ◽  
Pavel Marášek ◽  
Enrique Castaño ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Nucleolar Organizer ◽  
Rna Polymerase I ◽  
Nucleolar Organizer Regions ◽  
Polymerase I

scholarly journals Initiation of Nucleolar Assembly Is Independent of RNA Polymerase I Transcription

2000 ◽  
Vol 11 (8) ◽  
pp. 2705-2717 ◽  
Author(s):  
Thibaut Dousset ◽  
Chen Wang ◽  
Céline Verheggen ◽  
Danyang Chen ◽  
Danièle Hernandez-Verdun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Nucleolar Organizer ◽  
Rna Polymerase I ◽  
Nucleolar Organizer Regions ◽  
Rdna Transcription ◽  
M Phase ◽  
Upstream Binding Factor ◽  
Polymerase I ◽  
Processing Factors

This report examines the distribution of an RNA polymerase I transcription factor (upstream binding factor; UBF), pre-rRNA processing factors (nucleolin and fibrillarin), and pre-rRNAs throughout mitosis and postmitotic nucleologenesis in HeLa cells. The results demonstrate that nucleolin, fibrillarin, and pre-rRNAs synthesized at G2/M phase of the previous cell cycle are directly recruited to UBF-associated nucleolar organizer regions (NORs) early in telophase before chromosome decondensation. Unlike the fusion of prenucleolar bodies to the nucleoli, this early recruitment of processing factors and pre-rRNAs is independent of RNA polymerase I transcription. In the absence of polymerase I transcription, the initial localization of nucleolin, fibrillarin, and pre-rRNAs to UBF-associated NORs generates segregated mininucleoli that are similar to the larger ones observed in interphase cells grown under the same conditions. Pre-rRNAs are juxtaposed to UBF-nucleolin-fibrillarin caps that may represent the segregated nucleoli observed by electron microscopy. These findings lead to a revised model of nucleologenesis. We propose that nucleolar formation at the end of mitosis results from direct recruitment of processing factors and pre-rRNAs to UBF-associated NORs before or at the onset of rDNA transcription. This is followed by fusion of prepackaged prenucleolar bodies into the nucleolus. Pre-ribosomal ribonucleoproteins synthesized in the previous cell cycle may contribute to postmitotic nucleologenesis.

scholarly journals Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription

Chromosoma ◽  
1988 ◽  
Vol 96 (6) ◽  
pp. 411-416 ◽  
Author(s):  
Kathleen M. Rose ◽  
Jan Szopa ◽  
Fu-Sheng Han ◽  
Yung-Chi Cheng ◽  
Arndt Richter ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Gene Transcription ◽  
Topoisomerase I ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
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.

Identification of two steps during Xenopus ribosomal gene transcription that are sensitive to protein phosphorylation

1994 ◽  
Vol 14 (3) ◽  
pp. 2011-2020
Author(s):  
P Labhart
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase ◽  
Protein Phosphorylation ◽  
Gene Transcription ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Transcription Initiation ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Polymerase I

Protein kinase(s) and protein phosphatase(s) present in a Xenopus S-100 transcription extract strongly influence promoter-dependent transcription by RNA polymerase I. The protein kinase inhibitor 6-dimethyl-aminopurine causes transcription to increase, while the protein phosphatase inhibitor okadaic acid causes transcription to decrease. Repression is also observed with inhibitor 2, and the addition of extra protein phosphatase 1 stimulates transcription, indicating that the endogenous phosphatase is a type 1 enzyme. Partial fractionation of the system, single-round transcription reactions, and kinetic experiments show that two different steps during ribosomal gene transcription are sensitive to protein phosphorylation: okadaic acid affects a step before or during transcription initiation, while 6-dimethylaminopurine stimulates a process "late" in the reaction, possibly reinitiation. The present results are a clear demonstration that transcription by RNA polymerase I can be regulated by protein phosphorylation.

Enhancers for RNA polymerase I in mouse ribosomal DNA

1990 ◽  
Vol 10 (9) ◽  
pp. 4816-4825
Author(s):  
C S Pikaard ◽  
L K Pape ◽  
S L Henderson ◽  
K Ryan ◽  
M H Paalman ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Gene Promoter ◽  
Intergenic Spacer ◽  
Regulatory Elements ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Functional Consequences ◽  
In Trans ◽  
Polymerase I

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.

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