Tracking the Interactions of rRNA Processing Proteins during Nucleolar Assembly in Living Cells

Abstract

Reorganization of the nuclear machinery after mitosis is a fundamental but poorly understood process. Here, we investigate the recruitment of the nucleolar processing proteins in the nucleolus of living cells at the time of nucleus formation. We question the role of the prenucleolar bodies (PNBs), during migration of the processing proteins from the chromosome periphery to sites of rDNA transcription. Surprisingly, early and late processing proteins pass through the same PNBs as demonstrated by rapid two-color four-dimensional imaging and quantification, whereas a different order of processing protein recruitment into nucleoli is supported by differential sorting. Protein interactions along the recruitment pathway were investigated using a promising time-lapse analysis of fluorescence resonance energy transfer. For the first time, it was possible to detect in living cells the interactions between proteins of the same rRNA processing machinery in nucleoli. Interestingly interactions between such proteins also occur in PNBs but not at the chromosome periphery. The dynamics of these interactions suggests that PNBs are preassembly platforms for rRNA processing complexes. INTRODUCTIONIn higher eukaryotes, the nucleus is disassembled when chromosomes condense at the beginning of mitosis and reassemble at the end of mitosis. During mitosis, there is redistribution and/or inactivation of the nuclear machineries that will be further involved in rebuilding nuclear functions. One of the fundamental features of nuclear organization is that many components of the RNA synthesis and processing machineries are grouped into compartments (Spector, 1993;Lamond and Earnshaw, 1998;Matera, 1999;Gall, 2000;Misteli, 2000;Spector, 2001;Huang, 2002;Lamond and Spector, 2003). This implies that the recruitment of dedicated machineries and formation of discrete nuclear domains are crucial events at the beginning of interphase. In addition, recent data indicate that the entry as well as correct relocation of the nuclear machineries into newly forming nuclei is a step-dependent and ordered process (Savino et al., 2001;Prasanth et al., 2003;Bubulya et al., 2004;Leung et al., 2004). However, recruitment of the nucleolar processing proteins on transcription sites is still a poorly understood process. To better understand the rebuilding of nucleolar functions after mitosis, we chose to investigate the assembly of the nucleolar processing machinery in real time in living cells.The nucleolus is a functional compartment of the nucleus generated by ribosome biogenesis (Hadjiolov, 1985;Mélèse and Xue, 1995;Leung et al., 2003). It is also a plurifunctional domain involved in the three-dimensional (3D) organization of chromatin in the nucleus (Chubb et al., 2002), in the assembly of several ribonucleoprotein complexes (Pederson, 1998;Olson et al., 2000), and in the formation of nuclear speckles (Bubulya et al., 2004). The nucleolus therefore seems to be a key player of the nuclear functional architecture. Its functions depend on the activation and recru...

The traffic of proteins between nucleolar organizer regions and prenucleolar bodies governs the assembly of the nucleolus at exit of mitosis

“…The transfer lasts 15-20 minutes for early processing proteins localized in DFC (GFP-fibrillarin) while the recruitment of late processing proteins localized in GC (GFP-B23/NPM, GFP-Nop52 or GFP-Bop1) lasts at least 90 minutes. Remarkably, early and late proteins are in the same PNBs 16 but DFC proteins are recruited more rapidly than GC proteins indicating a differential sorting from PNBs and a different steady state in incipient nucleoli.…”

“…Permanently established HeLa cell lines expressing GFP-Nop52, GFP-B23, GFP-Bop and GFP-fibrillarin were already described. 14,16 The GFP-PP1γ cell line was a gift of Laura Trinckle. 24 The LMB (Calbiochem) stored at -80°C in small aliquots, was used at a concentration of 30 nM.…”

“…56 The full-length fibrillarin cDNA was a gift of J.P. Aris, and the fibrillarin-GFP and Nop52-GFP constructs have been described. 14 For colocalization, co-transfections with the constructs already described DsRed-B23, 56 DsRed-Nop52, 16 or mRFP-Nop56, 56 were used.…”

“…In parallel, prenucleolar bodies (PNBs) are assembled. [12][13][14][15][16] The PNBs contain nucleolar processing proteins, pre-rRNAs and snoRNAs. 17 Even though PNBs are formed in all animal and plant cells, their role in the dynamics of DFC and GC assembly is still unknown.…”

Bystin-like protein is upregulated in hepatocellular carcinoma and required for nucleologenesis in cancer cell proliferation

“…Some processing proteins transfer to nucleolus-derived foci (NDF) in the early anaphase cytoplasm [5]. During telophase, NDF disappear with a concomitant appearance of concentrated foci called prenucleolar bodies (PNBs) in the reforming nuclei [30][31][32][33]. Although it is not clear why intermediate steps including NDF and PNBs are required, formation of NDF and PNBs is crucial for nucleolar assembly.…”