Initiation of Nucleolar Assembly Is Independent of RNA Polymerase I Transcription

Abstract

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),...

Isolation and Proteomic Characterization of Human Parvulin-associating Preribosomal Ribonucleoprotein Complexes

“…Of these at least 3 proteins are reported or expected to be present in the nucleolus or nucleus. Among these the nucleolar protein includes the p160 Myb-binding protein (U63648) (43,54,55). The proteins known to be present in the nucleus include the homologue of targeting protein for Xenopus kinesin-like protein 2 (Xklp2) (TPX2, AF244547) (44 -46, 56 -58) and the p32cdc2-like PITSLRE protein kinase ␣ SV9 isoform (AF080683) (47,60).…”

Distinct genome protective vs. ribosome synthetic functions of the paralogous nucleolar proteins nucleostemin and GNL3L

“…However, as with short-term (2-day) nucleostemin knockdown, GNL3L depletion did not alter the fibrillarin-, nucleolin-or nucleosteminlabeled subnucleolar structures (Fig. 6C,D), indicating that the effect of the 2-day GNL3L knockdown on 47S-to-45S processing does not manifest as a change in nucleolar structure such as that known to occur when transcription of the rDNA is perturbed (Dousset et al, 2000;Ma and Pederson, 2013). …”

The traffic of proteins between nucleolar organizer regions and prenucleolar bodies governs the assembly of the nucleolus at exit of mitosis

“…On telophase cells progress to early G1a in 20 minutes before being arrested similarly to their neighbors, the arrested early G1a visible at the beginning of the time-lapse imaging. Together, these observations indicate that LMB-treated cells pass through mitosis, that nucleolar assembly is impaired after telophase and the cells are blocked in early G1a.Arrest of transcription could explain impairment of nucleolar assembly and maintenance of PNBs similarly to what happens when rDNA transcription is blocked by actinomycin D. 29 In interphase cells, 2 hours of LMB treatment have no effect on BrUTP incorporation in nucleoli (Fig. 5 Nu) and treatments prolonged for up to 6 hours do not induce nucleolar segregation, the typical reorganization process when rDNA transcription is blocked.…”

“…The pre-rRNAs in PNBs are synthesized during prophase and stabilized after one cleavage. 29 The link between nucleolar function and the presence of PNBs was established by inhibiting rDNA transcription or pre-rRNA processing while maintaining rDNA transcription. 15,29 In both cases, treatment with either actinomycin D or roscovitine, maintains the PNBs and impairs nucleolar assembly.…”

“…29 The link between nucleolar function and the presence of PNBs was established by inhibiting rDNA transcription or pre-rRNA processing while maintaining rDNA transcription. 15,29 In both cases, treatment with either actinomycin D or roscovitine, maintains the PNBs and impairs nucleolar assembly.We have observed here that the time of residency of GC molecules is higher in incipient than in interphase nucleoli (respectively t 1/2 of 31.7 seconds and 12.3 seconds). In incipient nucleoli the establishment of the vectorial process of ribosome assembly generated first DFC and then GC as demonstrated in several models.…”