Thanks to recent innovative methodologies, key cellular processes such as replication or transcription can be visualized directly in situ in intact tissues. Many studies use so-called click iT chemistry where nascent DNA can be tracked by 5-ethynyl-2'-deoxyuridine (EdU), and nascent RNA by 5-ethynyl uridine (EU). While the labeling of replicating DNA by EdU has already been well established and further exploited in plants, the use of EU to reveal nascent RNA has not been developed to such an extent. In this article, we present a protocol for labeling of nucleolar RNA transcripts using EU and show that EU effectively highlights the nucleolus. The method is advantageous, because the need to prepare transgenic plants expressing fluorescently tagged nucleolar components when the nucleolus has to be visualized can be avoided.

Institute of Biophysics Academy of Sciences of the Czech Republic Brno Czechia

Laboratory of Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czechia

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Brno Czechia

Zobrazit více v PubMed

Bernhard W. (1966). Ultrastructural aspects of the normal and pathological nucleolus in mammalian cells. Natl. Cancer Inst. Monogr. 23 13–38. PubMed

Beven A. F., Simpson G. G., Brown J. W., Shaw P. J. (1995). The organization of spliceosomal components in the nuclei of higher plants. J. Cell Sci. 108(Pt 2) 509–518. PubMed

Biggiogera M., Fakan S., Kaufmann S. H., Black A., Shaper J. H., Busch H. (1989). Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus. J. Histochem. Cytochem. 37 1371–1374. 10.1177/37.9.2768807 PubMed DOI

Casafont I., Navascues J., Pena E., Lafarga M., Berciano M. T. (2006). Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5’-fluorouridine into nascent RNA. Neuroscience 140 453–462. 10.1016/j.neuroscience.2006.02.030 PubMed DOI

Chandrasekhara C., Mohannath G., Blevins T., Pontvianne F., Pikaard C. S. (2016). Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis. Genes Dev. 30 177–190. 10.1101/gad.273755.115 PubMed DOI PMC

Chang W. Y., Winegarden N. A., Paraiso J. P., Stevens M. L., Westwood J. T. (2000). Visualization of nascent transcripts on Drosophila polytene chromosomes using BrUTP incorporation. Biotechniques 29 934–936. PubMed

Chen M., Jiang P. (2004). Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in HEp-2 cells. Acta Pharmacol. Sin. 25 902–906. PubMed

de Carcer G., Medina F. J. (1999). Simultaneous localization of transcription and early processing markers allows dissection of functional domains in the plant cell nucleolus. J. Struct. Biol. 128 139–151. 10.1006/jsbi.1999.4187 PubMed DOI

Dhoondia Z., Tarockoff R., Alhusini N., Medler S., Agarwal N., Ansari A. (2017). Analysis of termination of transcription using BrUTP-strand-specific transcription run-on (TRO) approach. J. Vis. Exp. 121:e55446. 10.3791/55446 PubMed DOI PMC

Dimitrova D. S. (2011). DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci. J. Cell Sci. 124(Pt 16) 2743–2752. 10.1242/jcs.082230 PubMed DOI

Dundr M., Raska I. (1993). Nonisotopic ultrastructural mapping of transcription sites within the nucleolus. Exp. Cell Res. 208 275–281. 10.1006/excr.1993.1247 PubMed DOI

Dvořáčková M., Raposo B., Matula P., Fuchs J., Schubert V., Peška V.et al. (2018). Replication of ribosomal DNA in Arabidopsis occurs both inside and outside of the nucleolus during S-phase progression. J. Cell Sci. 131:jcs.202416. 10.1242/jcs.202416 PubMed DOI

Fakan S., Hernandez-Verdun D. (1986). The nucleolus and the nucleolar organizer regions. Biol. Cell 56 189–205. 10.1111/j.1768-322X.1986.tb00452.x PubMed DOI

French S. L., Miller O. L., Jr. (1989). Transcription mapping of the Escherichia coli chromosome by electron microscopy. J. Bacteriol. 171 4207–4216. 10.1128/jb.171.8.4207-4216.1989 PubMed DOI PMC

Gratzner H. G. (1982). Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218 474–475. 10.1126/science.7123245 PubMed DOI

Hayashi K., Hasegawa J., Matsunaga S. (2013). The boundary of the meristematic and elongation zones in roots: endoreduplication precedes rapid cell expansion. Sci. Rep. 3:2723. 10.1038/srep02723 PubMed DOI PMC

Imamachi N., Tani H., Mizutani R., Imamura K., Irie T., Suzuki Y., et al. (2014). BRIC-seq: a genome-wide approach for determining RNA stability in mammalian cells. Methods 67 55–63. 10.1016/j.ymeth.2013.07.014 PubMed DOI

Jacob J., Sirlin J. L. (1964). Electron microscope autoradiography of the nucleolus of insect salivary gland cells. Nature 202 622–623. 10.1038/202622b0 PubMed DOI

Jao C. Y., Salic A. (2008). Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. U.S.A. 105 15779–15784. 10.1073/pnas.0808480105 PubMed DOI PMC

Jensen P. O., Larsen J., Larsen J. K. (1993). Flow cytometric measurement of RNA synthesis based on bromouridine labelling and combined with measurement of DNA content or cell surface antigen. Acta Oncol. 32 521–524. 10.3109/02841869309096111 PubMed DOI

Jordan E. G. (1984). Nucleolar nomenclature. J. Cell Sci. 67 217–220. PubMed

Jordan E. G., Luck B. T. (1976). The nucleolus organizer and the synaptonemal complex in Endymion non-scriptus (L.). J Cell Sci 22 75–86. PubMed

Kazda A., Akimcheva S., Watson J. M., Riha K. (2016). Cell proliferation analysis using EdU labeling in whole plant and histological samples of Arabidopsis. Methods Mol. Biol. 1370 169–182. 10.1007/978-1-4939-3142-2_13 PubMed DOI

Kennedy D. C., McKay C. S., Legault M. C., Danielson D. C., Blake J. A., Pegoraro A. F., et al. (2011). Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions. J. Am. Chem. Soc. 133 17993–18001. 10.1021/ja2083027 PubMed DOI

Koberna K., Malinsky J., Pliss A., Masata M., Vecerova J., Fialova M., et al. (2002). Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of “Christmas trees” in situ. J. Cell Biol. 157 743–748. 10.1083/jcb.200202007 PubMed DOI PMC

Kopecny V., Landa V., Malatesta M., Martin T. E., Fakan S. (1996). Immunoelectron microscope analyses of rat germinal vesicle-stage oocyte nucleolus-like bodies. Reprod. Nutr. Dev. 36 667–679. PubMed

Kotogany E., Dudits D., Horvath G. V., Ayaydin F. (2010). A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods 6:5. 10.1186/1746-4811-6-5 PubMed DOI PMC

Kramer B. (1980). The effect of actinomycin D on the nucleolus and on pigment synthesis in pigment cells of Xenopus laevis: an ultrastructural study. J. Anat. 130(Pt 4) 809–820. PubMed PMC

Kuang G. C., Michaels H. A., Simmons J. T., Clark R. J., Zhu L. (2010). Chelation-assisted, copper(II)-acetate-accelerated azide-alkyne cycloaddition. J. Org. Chem. 75 6540–6548. 10.1021/jo101305m PubMed DOI PMC

Mickelson-Young L., Wear E., Mulvaney P., Lee T. J., Szymanski E. S., Allen G., et al. (2016). A flow cytometric method for estimating S-phase duration in plants. J. Exp. Bot. 67 6077–6087. 10.1093/jxb/erw367 PubMed DOI PMC

Montanaro L., Govoni M., Orrico C., Trere D., Derenzini M. (2011). Location of rRNA transcription to the nucleolar components: disappearance of the fibrillar centers in nucleoli of regenerating rat hepatocytes. Cell Struct. Funct. 36 49–56. 10.1247/csf.10017 PubMed DOI

Ochs R. L., Lischwe M. A., Spohn W. H., Busch H. (1985). Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol. Cell 54 123–133. 10.1111/j.1768-322X.1985.tb00387.x PubMed DOI

Pendle A. F., Clark G. P., Boon R., Lewandowska D., Lam Y. W., Andersen J., et al. (2005). Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol. Biol. Cell 16 260–269. 10.1091/mbc.E04-09-0791 PubMed DOI PMC

Pontvianne F., Blevins T., Chandrasekhara C., Mozgova I., Hassel C., Pontes O. M., et al. (2013). Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes Dev. 27 1545–1550. 10.1101/gad.221648.113 PubMed DOI PMC

Pontvianne F., Matia I., Douet J., Tourmente S., Medina F. J., Echeverria M., et al. (2007). Characterization of AtNUC-L1 reveals a central role of nucleolin in nucleolus organization and silencing of AtNUC-L2 gene in Arabidopsis. Mol. Biol. Cell 18 369–379. 10.1091/mbc.E06-08-0751 PubMed DOI PMC

Pruitt R. E., Meyerowitz E. M. (1986). Characterization of the genome of Arabidopsis thaliana. J. Mol. Biol. 187 169–183. 10.1016/0022-2836(86)90226-3 PubMed DOI

Raska I., Reimer G., Jarník M., Kostrouch Z., Raska K., Jr (1989). Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components? Biol. Cell 65 79–82. PubMed

Raska I., Shaw P. J., Cmarko D. (2006). New insights into nucleolar architecture and activity. Int. Rev. Cytol. 255 177–235. 10.1016/S0074-7696(06)55004-1 PubMed DOI

Rostovtsev V. V., Green L. G., Fokin V. V., Sharpless K. B. (2002). A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41 2596–2599. 10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4 PubMed DOI

Scheer U., Xia B., Merkert H., Weisenberger D. (1997). Looking at Christmas trees in the nucleolus. Chromosoma 105 470–480. 10.1007/BF02510484 PubMed DOI

Shibasaki K., Uemura M., Tsurumi S., Rahman A. (2009). Auxin response in Arabidopsis under cold stress: underlying molecular mechanisms. Plant Cell 21 3823–3838. 10.1105/tpc.109.069906 PubMed DOI PMC

Singh B. N., Achary V. M. M., Panditi V., Sopory S. K., Reddy M. K. (2017). Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation. Plant Mol. Biol. 94 595–607. 10.1007/s11103-017-0626-4 PubMed DOI

So L. K., Cheung S. K., Ma H. L., Chen X. P., Cheng S. H., Lam Y. W. (2010). In situ labeling of transcription sites in marine medaka. J. Histochem. Cytochem. 58 173–181. 10.1369/jhc.2009.954511 PubMed DOI PMC

Stoykova A. S., Dabeva M. D., Dimova R. N., Hadjiolov A. A. (1985). Ribosome biogenesis and nucleolar ultrastructure in neuronal and oligodendroglial rat brain cells. J. Neurochem. 45 1667–1676. 10.1111/j.1471-4159.1985.tb10521.x PubMed DOI

Straatman K. H., Trompetter C. M., Schul W., Schel J. H. N. (1996). Fluorescent labelling of nascent RNA reveals nuclear transcription doamins throughout plant cell nuclei. Protoplasma 192 145–149. 10.1007/BF01273886 DOI

Tani H., Akimitsu N. (2012). Genome-wide technology for determining RNA stability in mammalian cells: historical perspective and recent advantages based on modified nucleotide labeling. RNA Biol. 9 1233–1238. 10.4161/rna.22036 PubMed DOI PMC

Thompson W. F., Beven A. F., Wells B., Shaw P. J. (1997). Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes. Plant J. 12 571–581. 10.1046/j.1365-313X.1997.00571.x PubMed DOI

Tornoe C. W., Christensen C., Meldal M. (2002). Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67 3057–3064. 10.1021/jo011148j PubMed DOI

Turner A. J., Knox A. A., Watkins N. J. (2012). Nucleolar disruption leads to the spatial separation of key 18S rRNA processing factors. RNA Biol. 9 175–186. 10.4161/rna.18811 PubMed DOI

Uddin M., Altmann G. G., Leblond C. P. (1984). Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine. J. Cell Biol. 98 1619–1629. 10.1083/jcb.98.5.1619 PubMed DOI PMC

Unuma T., Senda R., Muramatsu M. (1972). Nature of the so-called fibrillar component in the segregated nucleolus after treatment of actinomycin D. J. Electron Microsc. 21 60–70. PubMed

Uttamapinant C., Tangpeerachaikul A., Grecian S., Clarke S., Singh U., Slade P., et al. (2012). Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling. Angew. Chem. Int. Ed. 51 5852–5856. 10.1002/anie.201108181 PubMed DOI PMC

Waksmundzka M., Debey P. (2001). Electric field-mediated BrUTP uptake by mouse oocytes, eggs, and embryos. Mol. Reprod. Dev. 58 173–179. 10.1002/1098-2795(200102)58:2<173::AID-MRD6>3.0.CO;2-2 PubMed DOI

Wansink D. G., Schul W., van der Kraan I., van Steensel B., van Driel R., de Jong L. (1993). Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J. Cell Biol. 122 283–293. 10.1083/jcb.122.2.283 PubMed DOI PMC

Wassermann K., Newman R. A., Davis F. M., Mullins T. D., Rose K. M. (1988). Selective inhibition of human ribosomal gene transcription by the morpholinyl anthracyclines cyanomorpholinyl- and morpholinyldoxorubicin. Cancer Res. 48 4101–4106. PubMed

Watson J. M., Platzer A., Kazda A., Akimcheva S., Valuchova S., Nizhynska V., et al. (2016). Germline replications and somatic mutation accumulation are independent of vegetative life span in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 113 12226–12231. 10.1073/pnas.1609686113 PubMed DOI PMC

Yokoyama R., Hirakawa T., Hayashi S., Sakamoto T., Matsunaga S. (2016). Dynamics of plant DNA replication based on PCNA visualization. Sci. Rep. 6:29657. 10.1038/srep29657 PubMed DOI PMC

Yung B. Y., Bor A. M., Chan P. K. (1990). Short exposure to actinomycin D induces “reversible” translocation of protein B23 as well as “reversible” inhibition of cell growth and RNA synthesis in HeLa cells. Cancer Res. 50 5987–5991. PubMed

Zankl H., Bernhardt S. (1977). Combined silver staining of the nucleolus organizing regions and Giemsa banding in human chromosomes. Hum. Genet. 37 79–80. 10.1007/BF00293775 PubMed DOI

Zhang X. H., Yu X. Z., Yue D. M. (2016). Phytotoxicity of dimethyl sulfoxide (DMSO) to rice seedlings. Int. J. Environ. Sci. Technol. 13 607–614. 10.1007/s13762-015-0899-6 DOI

Insight into chromatin compaction and spatial organization in rice interphase nuclei

Visualization of the Nucleolus Using 5' Ethynyl Uridine