3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription

In each bead of the nucleolar necklace, using adenosine analog DRB-treated PtK1 cells, we investigated the three components of rDNA transcription, i.e . the gene, transcription factor UBF and transcripts. In situ hybridization revealed the unraveling and 3-D dispersion of most of the rDNA coding sequ ences within the nucleus. The signals were small, of similar intensity and tandemly organized in the necklace, This observation is compatible with the fact that they might correspond to single gene units. Active transcription was visualized in these units, demonstrating that they were active functio nal units. Transcript labeling was not similar for each unit, contrary to U BF labeling, UBF and rRNA transcripts were only partially colocalized, as d emonstrated by 3-D image analysis and quantification. As visualized by elec tron microscopy, the necklace was composed of a small fibrillar center part ially surrounded by a dense fibrillar component. The 3-D arrangement of thi s individual unit in the necklace, investigated both by confocal and electr on microscopy in the same cells, showed that the individual beads were link ed by a dense fibrillar component. The reversibility of this organization a fter removal of DRB indicated that the beads in the necklace are certainly the elementary functional domain of the nucleolus. In addition, these resul ts lead us to suggest that the organization of a functional domain, presuma bly corresponding to a single gene, can be studied by in situ approaches.