Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex.

To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p-Nup159p-Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

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.

Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae.

By combining cryofixation and cryosubstitution in a structural and functional analysis of the nucleus of Saccharomyces cerevisiae, we identified morphological subcompartments in the nucleolus. These were similar to those of nucleoli of higher eukaryotes, such as the fibrillar centre (FC), the dense fibrillar component (DFC) and the granular component (GC). In situ hybridization and immunocytochemistry revealed RNA polymerase I and proteins involved in early steps of ribosomal maturation along the DFC, while the ribosomal genes were detected at the FCs. Our results also suggest that ribosomal transcripts are distributed along a nucleolar network that might include both DFC and GC. We also show that pre-ribosomal subunits may be exported along tracks to the cytoplasm. Export takes place through all the pores of the nuclear envelope, not just those in contact with the nucleolus. Moreover, comparison of the nucleolar organization in S. cerevisiae and in Schizosaccharomyces pombe demonstrated than the distribution of the 5S genes with respect to the 35S transcription unit does not modify the organization of the nucleolus. We also report, for the first time, the ultrastructural localization of RNA polymerase II in yeast. The distribution of RNA polymerase II and morphological details that could be observed in the extra-nucleolar region of cryofixed cells provided cytological evidence of a peripheral region extending along the nuclear envelope that could correspond to heterochromatin in higher eukaryotes.

Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway.

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.

The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains.

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

Ultrastructural changes in the Schizosaccharomyces pombe nucleolus following the disruption of the gar2+ gene, which encodes a nucleolar protein structurally related to nucleolin.

The nucleolar protein gar2, from the fission yeast Schizosaccharomyces pombe, is the functional homolog of NSR1 from Saccharomyces cerevisiae, and is structurally related to nucleolin from vertebrates. By immunocytochemistry at the electron microscope level, we show that gar2 co-localizes with RNA polymerase I and the gar1 protein along the dense fibrillar component of the nucleolus in a wild-type strain of S. pombe, suggesting that gar2 is involved in the transcription and/or in the early steps of maturation of the ribosomal RNAs. Since the effects of disruption of the gar2+ gene might also shed light on the role of the gar2 protein, we analyzed the ultrastructure of the nucleolus of a gar2-disruption mutant. The nucleolus of the gar2- mutant is dramatically reorganized when compared with that of the wild-type gar2+ strain: a truncated protein containing the NH2-terminus of the gar2 protein is accumulated in an unusual nucleolar "dense body". Our results also suggest that the NH2-terminus might be sufficient for nucleolar localization via interaction with specific nucleolar components and support the hypothesis that gar2 in wild-type S. pombe interacts with nascent pre-rRNA via its two RNA-binding domains in combination with the glycine/arginine-rich domain. We also report that disruption of the gar2+ gene results in a mutant that is defective in cytokinesis and nuclear division.

Structural and functional analysis of the nucleolus of the fission yeast Schizosaccharomyces pombe.

Yeasts are an attractive model for the study of ribosome synthesis. However, our understanding of the relationship between the structure and function of the yeast nucleolus, in which preribosomal particles are synthesized, requires further investigations using microscopic approaches and in situ molecular biology. Combining cryofixation and cryosubstitution of Schizosaccharomyces pombe, we could identify morphologically distinct substructures in the nucleolus similar to the components of nucleoli of higher eukaryotes such as the fibrillar centers (FCs), the dense fibrillar component (DFC) and the granular component (GC). We complemented this morphological study by performing in situ hybridization and immunocytochemistry at the electron microscopy level. Using a probe complementary to the entire rRNA transcription unit of S. pombe, we detected rDNA at the periphery of the FCs, while immunocytochemistry with antibodies specific for the RNA polymerase I and the gar1 protein provided evidence that transcription and early steps of maturation take place in the DFC that extends throughout the nucleolus. We also present evidence that preribosomal subunits may be exported along tracks to the cytoplasm through all of the pores of the nuclear envelope and not just those in the portion of the envelope close to the nucleolus.