Spatial dynamics of rRNAs

Abstract

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 3H-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 1xVisualization of focal sites of transcription within human nuclei. Jackson, D.A et al. EMBO J. 1993; 12: 1054–1065See all References, 2xFluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. Wansink, D.G et al. J. Cell Biol. 1993; 122: 283–293Crossref | PubMedSee all References, 3xNonisotopic ultrastructural mapping of transcription sites within the nucleolus. Dundr, M and Raska, I. Exp. Cell Res. 1993; 208: 275–281Crossref | PubMedSee all References. 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 (4xSee all References4). By improving cell preservation in this manner, both extranucleolar RNA transcripts (sensitive to α-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 (Fig. 1Fig. 1). 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 5xMovement of nuclear poly(A)RNA throughout the interchromatin space in living cells. Politz, J.C et al. Curr. Biol. 1999; 9: 285–291Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all References5. 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.Fig. 1Localization of bromodeoxyuridine 5′-triphosphate (BrUTP)-labeled rRNAs during their movement from initial sites of transcription to the periphery of the nucleoli. HeLa cells, treated with α-amanitin to inhibit extranucleolar transcription, were transfected by lipofection for 15 min with BrUTP-FuGene-6 complexes and then cultured for various lengths of time before being fixed. Bromo-rRNAs were detected by secondary FITC-coupled antibodies. For each z series, 15-30 optical sections were recorded with a 0.25 μm z-step by exciting the fluorophore at 488 nm and by the simultaneous collection of phase-contrast images. Files were processed using the Analyze software. A surface-visualization procedure was used for each reconstructed volume 6xElectron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization. Heliot, L et al. Mol. Biol. Cell. 1998; 8: 2199–2216CrossrefSee all References6. Results show either a single optical section (a, c, e, g) or a three-dimensional reconstruction of the whole volume (b, d, f, h). Insets in (b), (d) and (f) show a higher magnification of three selected areas. Bar, 5 μm [1 μm for insets in (b) and (d), 2.5 μm for inset in (f)]. The nuclear limits, determined by phase contrast, are represented by dotted lines. A time-course of BrUTP incorporation appeared as a radial flow starting simultaneously from numerous transcription sites, then growing into concentric rings, which finally fused together as they progressed towards the nucleolar periphery.View Large Image | Download PowerPoint Slide