This report examines the distribution of an RNA polymerase I transcription factor (upstream binding factor; UBF), pre-rRNA processing factors (nucleolin and fibrillarin), and pre-rRNAs throughout mitosis and postmitotic nucleologenesis in HeLa cells. The results demonstrate that nucleolin, fibrillarin, and pre-rRNAs synthesized at G2/M phase of the previous cell cycle are directly recruited to UBF-associated nucleolar organizer regions (NORs) early in telophase before chromosome decondensation. Unlike the fusion of prenucleolar bodies to the nucleoli, this early recruitment of processing factors and pre-rRNAs is independent of RNA polymerase I transcription. In the absence of polymerase I transcription, the initial localization of nucleolin, fibrillarin, and pre-rRNAs to UBF-associated NORs generates segregated mininucleoli that are similar to the larger ones observed in interphase cells grown under the same conditions. Pre-rRNAs are juxtaposed to UBF-nucleolin-fibrillarin caps that may represent the segregated nucleoli observed by electron microscopy. These findings lead to a revised model of nucleologenesis. We propose that nucleolar formation at the end of mitosis results from direct recruitment of processing factors and pre-rRNAs to UBF-associated NORs before or at the onset of rDNA transcription. This is followed by fusion of prepackaged prenucleolar bodies into the nucleolus. Pre-ribosomal ribonucleoproteins synthesized in the previous cell cycle may contribute to postmitotic nucleologenesis. INTRODUCTIONThe nucleus is a complex structure within which various functions and components are spatially and temporally organized and interregulated (reviewed by Lamond and Earnshaw, 1998;Dreyfuss and Struhl, 1999). Nucleoli represent the most prominent example of this organization. The major function of the nucleolus is ribosome biogenesis (reviewed by Busch and Smetana, 1970). rRNAs are synthesized, processed, cleaved, and assembled with ribosomal proteins in the nucleolus before export to the cytoplasm. The nucleolus is composed of three structurally distinguishable constituents: fibrillar centers, dense fibrillar components, and granular components, as observed by electron microscopy (reviewed by Busch and Smetana, 1970;Hadjiolov, 1985). Although the spatial and temporal relationship between rRNA synthesis and the three structural constituents remains to be clarified, it is generally thought that transcription of rDNA takes place in a transient area between the fibrillar centers and the dense fibrillar components and that rRNA processing and preribosomal particle assembly progress from the dense fibrillar components to the surrounding granular components (reviewed by Spector et al., 1993;Shaw and Jordan, 1995;Scheer and Hock, 1999). More recent evidence (reviewed by Pederson, 1998;Garcia and Pillus, 1999) suggests that nucleoli have additional functions related to the cell cycle Visintin et al., 1999), cellular aging (Straight et al., 1999), signal recognition particle biosynthesis (Jacobson and Pederson, 1998),...