Abstract
This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5′-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in ∼10 d.
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Acknowledgements
We thank Miss F. Skivée for their skillful technical assistance. This work received financial support from the 'Fonds National de la Recherche Scientifique Médicale' (grant no. 3.4540.06), ARC (contract no. 4497) and Ligue contre le Cancer (Departements de l'Aube, de la Marne et des Ardennes). F.L. and N.T. are PhD grant holders of the FNRS.
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41596_2008_BFnprot2008198_MOESM352_ESM.mov
Supplementary Video 1: Volume rendering of Br-RNAs after a 15 min pulse within a multinucleated cell (without α-amanitin) showing a strong nucleolar, nucleoplasmic and cytoplasmic labelling. (MOV 2602 kb)
41596_2008_BFnprot2008198_MOESM353_ESM.mov
Supplementary Video 2: Volume rendering of Br-RNAs after a 15 min pulse within two α-amanitin-treated cells showing a strong nucleolar and cytoplasmic labelling. (MOV 1869 kb)
41596_2008_BFnprot2008198_MOESM354_ESM.mov
Supplementary Video 3: Surface rendering of Br-RNAs and RNA polymerase 1 after a 5 min pulse within a α-amanitin treated cell. Br-RNAs (solid green), RNA polymerase 1 (solid red) are shown alone or simultaneously on left, middle and right panels respectively. The two labellings appear as totally overlapping individual full beads 0.5 to 1 µm in diameter, organized as several necklaces. (MOV 2194 kb)
41596_2008_BFnprot2008198_MOESM355_ESM.mov
Supplementary Video 4: Surface rendering of Br-RNAs and RNA polymerase 1 after a 5 min pulse within a α-amanitin treated cell (same cell as on movie 3). Br-RNAs (transparent green) and RNA polymerase 1 (solid red) are shown simultaneously (left panel). Voxels with co-localized high levels of green and red signals were computed and shown in solid blue either alone (middle panel) or simultaneously with Br-RNAs (transparent green) (right panel). Note that co-localized voxels represent 95% of voxels containing high levels of BrRNA labelling and that they are located in its central part. (MOV 2709 kb)
41596_2008_BFnprot2008198_MOESM356_ESM.mov
Supplementary Video 5: Surface rendering of Br-RNAs and RNA polymerase 1 after a 15 min pulse within a α-amanitin treated cell. Br-RNAs (solid green), RNA polymerase 1 (solid red) are shown alone or simultaneously on left, middle and right panels respectively. Br-RNAs labelling is disposed as large hollow beads partly overlapping RNA polymerase 1 labelling. The latter is disposed as individual full beads 0.5 to 1 µm in diameter, organized as several necklaces. (MOV 2590 kb)
41596_2008_BFnprot2008198_MOESM357_ESM.mov
Supplementary Video 6: Surface rendering of Br-RNAs and RNA polymerase 1 after a 15 min pulse within a α-amanitin treated cell (same cell as on movie 5). Br-RNAs (transparent green) and RNA polymerase 1 (solid red) are shown simultaneously (left panel). Voxels with co-localized high levels of green and red signals were computed and shown in solid blue either alone (middle panel) or simultaneously with Br-RNAs (transparent green) (right panel). Note that co-localized voxels represent 69% of voxels containing high levels of BrRNA labelling and that they are located in its central part. (MOV 2827 kb)
41596_2008_BFnprot2008198_MOESM358_ESM.mov
Supplementary Video 7: Surface rendering of Br-RNAs and RNA polymerase 1 after a 30 min pulse within a α-amanitin treated cell. Br-RNAs (solid green), RNA polymerase 1 (solid red) are shown alone or simultaneously on left, middle and right panels respectively. Br-RNAs labelling constitutes large sponge-like structures with many holes. Some of the latter contain RNA polymerase 1 labelling which is disposed as individual full beads 0.5 to 1 µm in diameter and organized as several necklaces. (MOV 2785 kb)
41596_2008_BFnprot2008198_MOESM359_ESM.mov
Supplementary Video 8: Surface rendering of Br-RNAs and RNA polymerase 1 after a 30 min pulse within a α-amanitin treated cell (same cell as on movie 7). Br-RNAs (transparent green) and RNA polymerase 1 (solid red) are shown simultaneously (left panel). Voxels with co-localized high levels of green and red signals were computed and shown in solid blue either alone (middle panel) or simultaneously with Br-RNAs (transparent green) (right panel). Note that co-localized voxels represent only 15% of voxels containing high levels of BrRNA labelling. (MOV 2593 kb)
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Thiry, M., Lamaye, F., Thelen, N. et al. A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA. Nat Protoc 3, 1997–2004 (2008). https://doi.org/10.1038/nprot.2008.198
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DOI: https://doi.org/10.1038/nprot.2008.198
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