Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.     

Mapping and characterization of the functional domains of the nucleolar protein RNA helicase II/Gu

RNA helicase II/Gu (RH-II/Gu) is a nucleolar RNA helicase of the DEAD-box superfamily. In this study, the functional domains of RH-II/Gu molecule were mapped by fusing the protein or its deletion mutants with a green fluorescence protein and subsequently transfecting or microinjecting the recombinant constructs into HeLa cells. In addition to the identification of a nuclear localization signal (NLS) in the N-terminus and a nucleolar targeting signal in the central helicase domain, a hidden NLS and a nucleolar targeting signal were found in the C-terminal arginine/glycine-rich domain. RH-II/Gu colocalized with fibrillarin, a component of the dense fibrillar region of the nucleolus. Overexpression of the entire RH-II/Gu protein or specific domains of the protein in HeLa cells did not interfere with the normal distribution of fibrillarin. However, when the helicase domain was truncated, the distribution pattern of fibrillarin was distorted. Microinjection of the wild-type RH-II/Gu cDNA into the nucleus of HeLa cells did not disrupt normal cell growth. However, when cells were injected with mutant DNA, only a small percentage of HeLa cells progressed through the cell cycle. Analysis of centrosomes in transfected cells demonstrated that most of the mutant-expressing cells were arrested early in the cell cycle. The results suggest that each of the structural domains of RH-II/Gu is necessary for cell growth and cell cycle progression.     

In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA

In situ hybridization using biotinylated rDNA probes and secondary antibody coupled to gold particles was developed on ultrathin sections of Lowicryl-embedded Ehrlich tumor cells for precise localization of ribosomal RNA (rRNA) and ribosomal DNA (rDNA). For the detection of rDNA, an immunocytochemical approach involving an antibody against single-stranded DNA was used in order to determine the more efficient denaturation procedure. Using this technique, rDNA can be visualized in the fibrillar centers of nucleoli, especially in their peripheral regions at the proximity of both the dense fibrils and the nucleolar interstices as well as within the latter. rDNA was occasionally detected in some clumps of dense nucleolus-associated chromatin. Besides the presence of rRNA in the ribosome-rich cytoplasmic areas and in the dense fibrillar component and the granular component of the nucleolus, rRNA was also found in the fibrillar center areas close to the boundary region to the dense fibrillar component. These results are discussed in the light of the present knowledge on the functional organization of the nucleolus.     

Does the synthesis of ribosomal RNA take place-within nucleolar fibrillar centers or dense fibrillar components?

By means of immunocytochemistry performed on cryosections of cultured cells, RNA polymerase I was localized mainly to nucleolar fibrillar centers. The labelling of nucleolar dense fibrillar components was low and depended on the cell type. In contrast, DNA topoisomerase I and RNP complexes containing U3 snRNA were enriched in dense fibrillar components, their occurrence in fibrillar centers being usually much less.     

Localization of chromatin in "persistent" nucleoli in okadaic acid treated HeLa cells.

In okadaic acid treated HeLa cells, the chromosomes sometimes condense without being accompanied by nuclear envelope breakdown. These cells show "persistent" nucleoli. Within these "persistent" nucleoli the intranucleolar chromatin condenses and can be observed in the region of the dense nucleolar component (DNC) of the nucleoli. Other nucleolar components, namely the fibrillar centre (FC) and the granular component (GC) remain unchanged. These observations strongly speak for the localization of nucleolar chromatin (ribosomal cistrons) within the dense nucleolar component of the interphase nucleolus.     

Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis

The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I-specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.