Components of the Nucleolar Processing Complex (Pre-rRNA, Fibrillarin, and Nucleolin) Colocalize during Mitosis and Are Incorporated to Daughter Cell Nucleoli

Abstract

We have traced in onion root cells the mitotic course of rRNA, of the RNA synthesized in the G2 period of the preceding interphase, and of the nucleolar proteins fibrillarin and nucleolin. The rRNA was detected by ultrastructural in situ hybridization with a rDNA probe capable of hybridizing mature rRNAs and also the intermediate forms of pre-rRNA processing. The RNA synthesized in the preceding G2 (which, according to classical data, is mostly rRNA) was revealed by autoradiography on synchronous cells labeled in G2 by tritiated uridine. Fibrillarin was detected by immunofluorescence in both mammalian and onion cells; the results in the latter cells were compared with those obtained after AgNOR staining. Electron microscopical immunocytochemistry was used to detect fibrillarin and nucleolin in onion cells. In all cases, following nucleolar dispersion in prophase, the signal was detected in the chromosome periphery during metaphase and anaphase, in irregular fibrillar masses located between chromosomes in ana-telophase, in prenucleolar bodies during telophase, and in the newly formed nucleoli, after nucleologenesis. Moreover, as expected, ribosomes appeared labeled after in situ hybridization, but a dispersed cytoplasmic labeling was observed in all experiments, mainly during metaphase and anaphase. These results demonstrate that nucleolar components involved in pre-rRNA processing, including rRNA itself, probably in an incompletely processed form, are transferred from the parental to the daughter cell nucleoli by means of transient structures, such as the perichromosomal sheath and prenucleolar bodies. Since these macromolecular components are assembled in the interphase nucleolus, forming the RNP processing complex, their colocalization during mitosis in the same transient structures strongly suggests that at least a subset of these complexes does not disaggregate during cell division, but remains assembled and becomes incorporated to the new nucleolus. Therefore, ribosome biogenesis restarts not only after mitosis at the level of transcription, but also at the intermediate levels of pre-rRNA processing.