Spatial dynamics of rRNAs

The understanding of the functional organization of a compartmentalized organelle such as the nucleus constitutes one of the major challenges in cell biology today. An essential step towards this goal is the study of the spatial dynamics of RNA synthesis and maturation. The in vivo dynamics of nascent RNAs have been commonly investigated by the labeling of transcripts with super(3)H-uridine, followed by autoradiographic detection. Because of the drawbacks inherent to this technique, an immunocytochemical technique, using bromodeoxyuridine 5'-triphosphate (BrUTP) as an analog, has been developed. However, cell permeabilization or microinjection techniques, which are employed to ensure the intracellular penetration of BrdU, disturb cell integrity. Consequently, although this technique has allowed the visualization of nucleolar labeling in the vicinity of the polymerase, it has not permitted the observation of nascent rRNA transport. To improve this approach, we have used the FuGene-6 transfection reagent for the introduction of BrUTP. By improving cell preservation in this manner, both extranucleolar RNA transcripts (sensitive to alpha -amanitin) and ribosomal RNAs (sensitive to actinomycin D) have been rapidly and simultaneously detected. Furthermore, the transport of nascent RNAs was visualized both within the nucleus and from the nucleus to the cytoplasm. By using pulse-chase experiments, three-dimensional reconstructions from confocal sections, and electron microscopy analysis of ultrathin sections, the topological and spatial dynamics of rRNAs within the nucleolus were revealed. Our data demonstrate that rRNA transport does not involve tracks. By contrast, it follows a volumic, well-defined pathway from multiple points that extend from nucleolar speckles towards the periphery of the nucleolus. This migration can be summarized by four typical labeling patterns. As assessed by double-labeling experiments with nucleolar proteins involved in rRNA synthesis or maturation, these patterns could correspond to different functional domains. The dynamics of rRNAs within the nucleolus are similar to those of some polyadenylated mRNAs within the extranucleolar space. Our BrUTP incorporation technique, combined with three-dimensional visualizations and tomographic analyses, should aid significantly our understanding of the functional, volumic organization of the cell nucleus.