Assembly and functional organization of the nucleolus: ultrastructural analysis of Saccharomyces cerevisiae mutants.
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.
Trumtel
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S
Laboratoire de Biologie Moléculaire Eucaryote, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5099, and Université Paul Sabatier, 31062 Toulouse Cedex, France.
Léger-Silvestre
I
I
Gleizes
P E
PE
Teulières
F
F
Gas
N
N
D001426:Bacterial Proteins; D001665:Binding Sites; D002466:Cell Nucleolus / Q000648:ultrastructure*; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D005656:Fungal Proteins / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D009697:Nucleolus Organizer Region / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D010957:Plasmids; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D020718:Ribonucleoproteins, Small Nucleolar; D012269:Ribosomal Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D005656:Fungal Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D020718:Ribonucleoproteins, Small Nucleolar; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C048502:ribosomal protein SS1, Streptomyces aureofaciens; C073180:GAR1 protein, S cerevisiae