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. 2007 May 21;177(4):573-8.
doi: 10.1083/jcb.200612048.

Intranucleolar sites of ribosome biogenesis defined by the localization of early binding ribosomal proteins

Tim Krüger  1 , Hanswalter Zentgraf, Ulrich Scheer
Affiliations

Intranucleolar sites of ribosome biogenesis defined by the localization of early binding ribosomal proteins

Tim Krüger et al. J Cell Biol. .

Abstract

Considerable efforts are being undertaken to elucidate the processes of ribosome biogenesis. Although various preribosomal RNP complexes have been isolated and molecularly characterized, the order of ribosomal protein (r-protein) addition to the emerging ribosome subunits is largely unknown. Furthermore, the correlation between the ribosome assembly pathway and the structural organization of the dedicated ribosome factory, the nucleolus, is not well established. We have analyzed the nucleolar localization of several early binding r-proteins in human cells, applying various methods, including live-cell imaging and electron microscopy. We have located all examined r-proteins (S4, S6, S7, S9, S14, and L4) in the granular component (GC), which is the nucleolar region where later pre-ribosomal RNA (rRNA) processing steps take place. These results imply that early binding r-proteins do not assemble with nascent pre-rRNA transcripts in the dense fibrillar component (DFC), as is generally believed, and provide a link between r-protein assembly and the emergence of distinct granules at the DFC-GC interface.

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Figures

Figure 1.
Figure 1.
Localization of early binding r-proteins in human Hep2 cells by confocal microscopy after transient expression as fusions with fluorescent proteins or with a myc epitope. (a–h) The distribution of r-proteins fused to GFP or the rapidly maturing spectral variant Venus is shown in living cells, whereas myc-tagged r-proteins were visualized by immunofluorescence of fixed cells with anti-myc antibodies. Note the characteristic distribution pattern of all expressed r-proteins, which are concentrated in the nucleoli, almost absent from the nucleoplasm, and dispersed throughout the cytoplasm. (rows i and j) L4 and S4 do not colocalize with mRFP-tagged fibrillarin, a marker of the DFC of the nucleolus, as revealed by double expression in live Hep2 cells. Note that the fibrillarin-positive structures (i′ and j′) correspond to regions deficient of r-proteins (i and j; the merged images are shown in i″ and j″). In contrast, L4-GFP colocalizes with B23-mRFP, which marks the GC (row i, insets). Corresponding differential interference contrast (DIC) images are also shown. Bars, 10 μm.
Figure 2.
Figure 2.
Characterization of mAb S14-39 raised against recombinant S14. (a) The antibodies stain the nucleolus and the cytoplasm of Hep2 cells. (b) In immunoblots, the mAb recognizes a 15-kD polypeptide of the small (40S) ribosomal subunit. (c) Incorporation of S4-GFP into cytoplasmic ribosomes. Ribosomes were isolated from a cell line stably expressing S4-GFP, and the subunits were analyzed by SDS-PAGE. Antibodies against GFP recognize an ∼55-kD band of the small subunit, the expected molecular mass of the fusion protein (left). The separation of small and large ribosomal subunits was monitored with antibodies to L10 (right). Bar, 10 μm.
Figure 3.
Figure 3.
Early binding r-proteins are restricted to the GC and absent from the DFC of nucleoli. After AMD treatment, the two major nucleolar components, GC and DFC, segregate into separate structures (a″, inset; coexpression of the GC marker B23-GFP and the DFC marker fibrillarin-RFP). Endogenous S14 is absent from the DFC, stained with antibodies to fibrillarin (a–a″). S6-GFP is also absent from the DFC, marked by expression of fibrillarin-RFP in living cells (b–b″). DRB causes the unraveling of nucleoli into DFC (stained with antibodies to fibrillarin; c′ and d′) and spherical GC remnants. S6 and L4 are associated exclusively with these GC structures (c and d; merged images in c″ and d″). (e–e″) Localization of S4-GFP and fibrillarin-mRFP in a live mitotic Hep2 cell undergoing nucleolar reformation. Shown is a telophase ∼20 min after the onset of anaphase. Reformation of the DFC around the chromosomal NORs occurs in the absence of S4. Around 10 min later, S4 begins to accumulate at the NORs, concurrent with the formation of the GC. Bars, 10 μm.
Figure 4.
Figure 4.
Immunogold electron microscopy showing the intranucleolar distribution of early binding r-proteins in Hep2 cells. FCs with the surrounding electron-dense DFC layer are clearly identified in all figures. Silver-enhanced gold particles are dispersed throughout the GC but are excluded from the DFC–FC complex. Myc-tagged S9, S14, and L4 were expressed in Hep2 cells and visualized using preembedding (b and c) or postembedding (a and d) labeling protocols followed by silver enhancement of the ultrasmall gold particles. Endogenous S14 was detected with mAb S14-39 (preembedding protocol; e). The DFC is accessible to antibodies, as illustrated by its strong labeling with antibodies to fibrillarin (mAb 72B9, preembedding protocol; f). Note that in this specific case, the silver enhancement reaction has been deliberately increased for optimized display of the signal in this survey image. The FCs are labeled with antibodies against RNA polymerase I (g). Bars, 0.2 μm.
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