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

Although rRNA synthesis, maturation, and assembly into preribosomal particl es occur within the nucleolus, the route taken by pre-rRNAs from their synt hetic sites toward the cytoplasm remains largely unexplored. Here, we emplo yed a nondestructive method for the incorporation of BrUTP into the RNA of living cells. By using pulse-chase experiments, three-dimensional image rec onstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we were able to describe topological and spatial dy namics of rRNAs within the nucleolus. We identified the precise location an d the volumic organization of four typical subdomains, in which rRNAs are s uccessively moving towards the nucleolar periphery during their synthesis a nd processing steps. The incorporation of BrUTP takes place simultaneously within several tiny spheres, centered on the fibrillar centers. Then, the s tructures containing the newly synthesized RNAs enlarge and appear as compa ct ringlets disposed around the fibrillar centers. Later, they form hollow spheres surrounding the latter components and begin to fuse together. Final ly, 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 starti ng from the fibrillar centers that form concentric rings, which finally fus e together as they progress toward the nucleolar periphery.