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.

scholarly journals In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

1996 ◽  
Vol 133 (2) ◽  
pp. 225-234 ◽  
Author(s):  
P Jordan ◽  
M Mannervik ◽  
L Tora ◽  
M Carmo-Fonseca
Keyword(s):  
Rna Polymerase ◽  
Actinomycin D ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Transcription ◽  
Polymerase I

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

scholarly journals Compositional reorganization of the nucleolus in budding yeast mitosis

2019 ◽  
Vol 30 (5) ◽  
pp. 591-606 ◽  
Author(s):  
Philipp Girke ◽  
Wolfgang Seufert
Keyword(s):  
Rna Polymerase ◽  
Budding Yeast ◽  
Protein Composition ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rdna Transcription ◽  
Unequal Distribution ◽  
Polymerase I ◽  
Nucleolar Segregation

The nucleolus is a membraneless organelle of the nucleus and the site of rRNA synthesis, maturation, and assembly into preribosomal particles. The nucleolus, organized around arrays of rRNA genes (rDNA), dissolves during prophase of mitosis in metazoans, when rDNA transcription ceases, and reforms in telophase, when rDNA transcription resumes. No such dissolution and reformation cycle exists in budding yeast, and the precise course of nucleolar segregation remains unclear. By quantitative live-cell imaging, we observed that the yeast nucleolus is reorganized in its protein composition during mitosis. Daughter cells received equal shares of preinitiation factors, which bind the RNA polymerase I promoter and the rDNA binding barrier protein Fob1, but only about one-third of RNA polymerase I and the processing factors Nop56 and Nsr1. The distribution bias was diminished in nonpolar chromosome segregation events observable in dyn1 mutants. Unequal distribution, however, was enhanced by defects in RNA polymerase I, suggesting that rDNA transcription supports nucleolar segregation. Indeed, quantification of pre-rRNA levels indicated ongoing rDNA transcription in yeast mitosis. These data, together with photobleaching experiments to measure nucleolar protein dynamics in anaphase, consolidate a model that explains the differential partitioning of nucleolar components in budding yeast mitosis.

scholarly journals Human Nopp140, Which Interacts with RNA Polymerase I: Implications for rRNA Gene Transcription and Nucleolar Structural Organization

1999 ◽  
Vol 19 (12) ◽  
pp. 8536-8546 ◽  
Author(s):  
Hung-Kai Chen ◽  
Chi-Yun Pai ◽  
Jing-Yi Huang ◽  
Ning-Hsing Yeh
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Gene Transcription ◽  
Ectopic Expression ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Amino Terminal ◽  
Polymerase I

ABSTRACT Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

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.

scholarly journals Migration of rat RNA polymerase I into chick erythrocyte nuclei undergoing reactivation in chick-rat heterokaryons.

1983 ◽  
Vol 97 (5) ◽  
pp. 1641-1643 ◽  
Author(s):  
U Scheer ◽  
G Lanfranchi ◽  
K M Rose ◽  
W W Franke ◽  
N R Ringertz
Keyword(s):  
Rna Polymerase ◽  
Immunofluorescence Microscopy ◽  
Sendai Virus ◽  
Specific Protein ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Time Interval ◽  
Reactivation Process ◽  
Polymerase I

Transcriptionally inactive chick erythrocyte nuclei were reactivated by Sendai virus-induced fusion of erythrocytes with rat L6J1 myoblasts. We used antibodies to trace the appearance of a specific protein engaged in transcription of a defined class of genes, those coding for rRNA, during reactivation. Using immunofluorescence microscopy, we found increasing amounts of rat RNA polymerase I to appear, during a certain period of time after fusion, in the reforming nucleoli of the chick nuclei. Amounts of rat RNA polymerase I sufficient to be detected by immunofluorescence microscopy had accumulated in the newly developed chick nucleoli 72-190 h after fusion was initiated. This time interval coincides with the time when chick rRNA synthesis can first be detected. The results raise the possibility that during these stages of the reactivation process chick rRNA genes are transcribed by heterologous RNA polymerase I molecules of rat origin.

scholarly journals I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

1998 ◽  
Vol 18 (10) ◽  
pp. 5809-5817 ◽  
Author(s):  
Jue Lin ◽  
Volker M. Vogt
Keyword(s):  
Rna Polymerase ◽  
Homing Endonuclease ◽  
Group I Intron ◽  
Subcellular Fractionation ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Temperature Sensitive ◽  
Group I ◽  
Polymerase I ◽  
Mutant Forms

ABSTRACT PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023–1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as theTetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.

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