Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae

Abstract

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.

Integrase Mediates Nuclear Localization of Ty3

“…Previous studies have indicated that pre-tRNAs and processing complexes may be distributed between nucleolar and nonnucleolar loci (25,45). However, electron microscopic localization of a polymerase III subunit in S. cerevisiae failed to find a significant concentration of the protein in the nucleolus, suggesting that the majority of polymerase III is not nucleolar (32). In this study we also did not find evidence that tRNA genes, the previously identified major chromosomal target of Ty3 integration, are generally localized to the nucleolus.…”

A cotranscriptional model for 3′-end processing of the Saccharomyces cerevisiae pre-ribosomal RNA precursor

“…Several studies have suggested a direct link between other pre-rRNA processing factors and RNA polymerase I. The U14 and U3 snoRNAs and the H/ACA Cotranscriptional rRNA processing www.rnajournal.org snoRNP protein Gar1p localize in the dense fibrillar component (Lazdins et al 1997;Leger-Silvestre et al 1999), the probable site of transcription by RNA polymerase I (Hozak et al 1994;Jackson and Cook 1995;Lazdins et al 1997;Mosgoeller et al 1998). In addition, the nucleolar processing factor Nop1p colocalizes with RNA polymerase I in mouse embryos (Cuadros-Fernandez and Esponda 1996) and with nascent rRNA transcripts (Garcia-Blanco et al 1995).…”

Diffusion-based Transport of Nascent Ribosomes in the Nucleus

“…The nuclear export of both the large and small ribosomal subunits has been shown to be dependent, at least indirectly, on the Ran-GTPase cycle and the exportin CRM1, and it is likely that CRM1-mediated export of 60S subunits requires the adaptor protein NMD3 (Moy and Silver, 1999;Ho et al, 2000;Gadal et al, 2001;Thomas and Kutay, 2003;Trotta et al, 2003). In situ hybridization experiments in fission yeast have indicated that rRNA may accumulate along short tracks when near the nuclear pores but nonetheless appears to exit from all the pores, not just those near the nucleolus (Léger-Silvestre et al, 1999). In situ hybridization studies in mammalian cells have primarily addressed the distribution of rRNA within the nucleolus (Huang, 2002) rather than the routes of extranucleolar rRNA traffic, because although high signal representing rRNA is routinely detected in both the nucleolus and the cytoplasm, nucleoplasmic signal which might represent ribosomes moving to the nuclear periphery is not easily detectable by in situ hybridization (PuvionDutilleul et al, 1991;Lazdins et al, 1997; J.C.R.…”