Three-dimensional organization of active rRNA genes within the nucleolus.
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.
Unité MéDIAN, CNRS UMR 6142, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France.
Thierry
T
Cheutin
Marie-Françoise
MF
O'Donohue
Adrien
A
Beorchia
Marc
M
Vandelaer
Hervé
H
Kaplan
Bruno
B
Deféver
Dominique
D
Ploton
Marc
M
Thiry
D000970:Antineoplastic Agents / Q000378:metabolism; D002460:Cell Line; D002466:Cell Nucleolus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D003609:Dactinomycin / Q000378:metabolism; D003839:Deoxyadenosines / Q000378:metabolism; D020459:Genes, rRNA; D006801:Humans; D007150:Immunohistochemistry; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D012318:RNA Polymerase I / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
D000970:Antineoplastic Agents; D004275:DNA, Ribosomal; D003839:Deoxyadenosines; D019384:Nucleic Acid Synthesis Inhibitors; D012335:RNA, Ribosomal; D003609:Dactinomycin; D012318:RNA Polymerase I; C058120:cordycepin; D014544:Uridine Triphosphate