Nucleolar organization of HeLa cells as studied by in situ hybridization

The distribution of the ribosomal genes and their ribosomal RNA (rRNA) products in the different compartments of the nucleolus of HeLa cells was examined on thin sections of Lowicryl embedded material. The ribosomal nucleic acids were visualized after hybridization with a set of biotinylated double-stranded ribosomal DNA (rDNA) probes from different locations along the gene, followed by immunogold labelling of biotin. Ribosomal genes were detected over both the entire fibrillar centres (FCs) and some masses of intranucleolar condensed chromatin. As for the rRNA components, comparison of the signal levels obtained with the different probes provides some information about the compartmentalization of distinct stages of ribosome biogenesis. Thus a probe specific for the 5' external transcribed spacer (5'ETS) portion of pre-rRNA labels almost exclusively the dense fibrillar component (DFC) and the border of the FCs, while the interior of the FCs appears devoid of any kind of rRNA species. By contrast, probes recognizing either 18S or 28S mature rRNA sequences label both the DFC and the granular component (GC). Moreover, mature 18S rRNA sequences are markedly under-represented relative to mature 28S rRNA sequences in the GC, as compared with the other nucleolar compartments. Our observations are consistent with the view that DFCs contain elongating and 47S-45S precursor rRNA molecules whereas the subsequent various rRNA processing intermediates are mainly located within the GC. Since the border of FCs is the only site where both rDNA and newly synthesized pre-rRNA coexist, the transcription of ribosomal genes seems to take place at the periphery of the FCs, and not in the DFC, suggesting that elongating and newly completed transcripts are immediately transferred into the surrounding DFC where they transiently accumulate before undergoing processing reactions and transfer to the GC.