Analysis of nucleolar pre-rRNA processing sites in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

The location of rRNA processing was analyzed by usingin situ hybridization with ITS1 probe and immunolabeling of anti-fibrillarin mAb in pea (Pisum sativum) root pole cells. The results showed that rRNA processing sites were in dense fibrillar components (DFCs) and granular components (GCs), but not in fibrillar centers (FCs). Low doses of actinomycin D (AMD) treatment can selectively suppress pre-rRNA synthesis but cannot disturb the processing of preformed pre-rRNAs. With AMD treatment prolonged, the density of labeled signals gradually decreased, indicating the preformed pre-rRNAs were gradually processed.     

Fibrillar nucleolar remnants do not contain macromolecular precursors of ribosomal RNA : Demonstration by the effects of -galactosamine

Administration of -galactosamine to rats produces inhibition of liver nuclear RNA synthesis and associated alterations in the structure of the nucleolus. Polyacrylamide gel electrophoretic analysis of liver nuclear RNA from galactosamine-treated rats has shown the virtual complete absence of ribosomal RNA (rRNA) precursor molecules at a time when the nucleolus consists solely of a dense fibrillar core devoid of granules. No evidence for an artefactual, preferential breakdown of nuclear RNA during extraction could be obtained from either 2.7 or 8% acrylamide gels. Furthermore, the almost complete cessation of nuclear RNA synthesis makes the possibility of there being rapid synthesis and degradation of ribosomal precursor molecules in vivo unlikely. With toluidine blue stains for RNA with nuclei isolated from galactosamine-treated animals, the large, brightly staining area associated with the normal nucleolus was not seen. On the basis of these observations, it is concluded that an RNA-depleted nucleolus appears fibrillar. It is suggested that the fibrillar material of a normal nucleolus may not itself be RNA even though this region does contain RNA precursor molecules.     

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus

Although rRNA synthesis, maturation, and assembly into preribosomal particles occur within the nucleolus, the route taken by pre-rRNAs from their synthetic sites toward the cytoplasm remains largely unexplored. Here, we employed a nondestructive method for the incorporation of BrUTP into the RNA of living cells. By using pulse-chase experiments, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we were able to describe topological and spatial dynamics of rRNAs within the nucleolus. We identified the precise location and the volumic organization of four typical subdomains, in which rRNAs are successively moving towards the nucleolar periphery during their synthesis and processing steps. The incorporation of BrUTP takes place simultaneously within several tiny spheres, centered on the fibrillar centers. Then, the structures containing the newly synthesized RNAs enlarge and appear as compact ringlets disposed around the fibrillar centers. Later, they form hollow spheres surrounding the latter components and begin to fuse together. Finally, these structures widen and form large rings reaching the limits of the nucleoli. These results clearly show that the transport of pre-rRNAs within the nucleolus does not occur randomly, but appears as a radial flow starting from the fibrillar centers that form concentric rings, which finally fuse together as they progress toward the nucleolar periphery.     

New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry.

We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.     

5'ETS rRNA processing facilitated by four small RNAs: U14, E3, U17, and U3.

The nucleolus, the compartment in which the large ribosomal RNA precursor (pre-rRNA) is synthesized, processed through a series of nucleolytic cleavages and modifications into the mature 18S, 5.8S, and 28S rRNAs, and assembled with proteins to form ribosomal subunits, also contains many small nucleolar RNAs (snoRNAs). We present evidence that the first processing event in mouse rRNA maturation, cleavage within the 5' external transcribed spacer, is facilitated by at least four snoRNAs: U14, U17(E1), and E3, as well as U3. These snoRNAs do not augment this processing by directing 2'-O-methylation of the pre-rRNA. A macromolecular complex in which this 5'ETS processing occurs may then function in the processing of 18S rRNA.     

Intranucleolar localization of the RNA polymerase A activity in isolated nuclei of regenerating rat liver

Ultrastructural autoradiography was used to visualize RNA polymerase A activity in parenchymal cell nuclei isolated from normal and regenerating (3, 24, 36 and 48 h after partial hepatectomy) rat liver. High resolution autoradiography showed that the activity of RNA polymerase A which was not inhibited by α-amanitin in a concentration of 0.8 μg/ml, was restricted to the nucleolus. Both the distribution pattern and number of grains were similar in control liver and regenerating liver 3 h after hepatectomy. Twentyfour, 36, and 48 h after hepatectomy nucleoli were enlarged and labeling was distinctly increased. In all experimental groups the activity of RNA polymerase A was located within fibrillar components of the nucleolus. The association of enzyme activity with this component was especially distinct in later stages (36 and 48 h) of liver regeneration. These results suggest that the fibrillar component of the nucleolus contains the active template for ribosomal RNA (rRNA) synthesis in rat liver cell nuclei.