Nucleolar assembly of the rRNA processing machinery in living cells

To understand how nuclear machineries are targeted to accurate locations du ring nuclear assembly. we investigated the pathway of the ribosomal RNA (rR NA) processing machinery towards ribosomal genes (nucleolar organizer regio ns [NORs]) at exit of mitosis. To follow in living cells two permanently tr ansfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In ea rly telophase, fibrillarin is concentrated simultaneously in prenucleolar b odies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These dist inct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a direc tional flow between PNBs and between PNBs and the nucleolus, ensuring progr essive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, sug gesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven b y pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formati on of one functional domain by cooperative interactions between different c hromosome territories.