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Metabolic Labeling in the Study of Mammalian Ribosomal RNA Synthesis

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Book cover The Nucleolus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1455))

Abstract

RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates.

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References

  1. Moss T, Langlois F, Gagnon-Kugler T, Stefanovsky V (2007) A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis. Cell Mol Life Sci 64(1):29–49

    Article  CAS  PubMed  Google Scholar 

  2. Stefanovsky VY, Pelletier G, Hannan R, Gagnon-Kugler T, Rothblum LI, Moss T (2001) An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF. Mol Cell 8(5):1063–1073

    Article  CAS  PubMed  Google Scholar 

  3. Stefanovsky V, Langlois F, Gagnon-Kugler T, Rothblum LI, Moss T (2006) Growth factor signaling regulates elongation of RNA polymerase I transcription in mammals via UBF phosphorylation and r-chromatin remodeling. Mol Cell 21(5):629–639. doi:10.1016/j.molcel.2006.01.023

    Article  CAS  PubMed  Google Scholar 

  4. Zhao J, Yuan X, Frodin M, Grummt I (2003) ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth. Mol Cell 11(2):405–413

    Article  CAS  PubMed  Google Scholar 

  5. Murayama A, Ohmori K, Fujimura A, Minami H, Yasuzawa-Tanaka K, Kuroda T, Oie S, Daitoku H, Okuwaki M, Nagata K, Fukamizu A, Kimura K, Shimizu T, Yanagisawa J (2008) Epigenetic control of rDNA loci in response to intracellular energy status. Cell 133(4):627–639. doi:10.1016/j.cell.2008.03.030, S0092-8674(08)00459-5 [pii]

    Article  CAS  PubMed  Google Scholar 

  6. Gagnon-Kugler T, Langlois F, Stefanovsky V, Lessard F, Moss T (2009) Loss of human ribosomal gene CpG methylation enhances cryptic RNA polymerase II transcription and disrupts ribosomal RNA processing. Mol Cell 35(4):414–425. doi:10.1016/j.molcel.2009.07.008, S1097-2765(09)00503-6 [pii]

    Article  CAS  PubMed  Google Scholar 

  7. Scherrer K, Latham H, Darnell JE (1963) Demonstration of an unstable RNA and of a precursor to ribosomal RNA in HeLa cells. Proc Natl Acad Sci U S A 49:240–248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Warner JR, Soeiro R, Birnboim HC, Girard M, Darnell JE (1966) Rapidly labeled HeLa cell nuclear RNA. I. Identification by zone sedimentation of a heterogeneous fraction separate from ribosomal precursor RNA. J Mol Biol 19(2):349–361

    Article  CAS  PubMed  Google Scholar 

  9. Loening UE, Jones KW, Birnstiel ML (1969) Properties of the ribosomal RNA precursor in Xenopus laevis; comparison to the precursor in mammals and in plants. J Mol Biol 45(2):353–366, 0022-2836(69)90110-7 [pii]

    Article  CAS  PubMed  Google Scholar 

  10. Hamdane N, Stefanovsky VY, Tremblay MG, Nemeth A, Paquet E, Lessard F, Sanij E, Hannan R, Moss T (2014) Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body. PLoS Genet 10(8):e1004505. doi:10.1371/journal.pgen.1004505

    Article  PubMed  PubMed Central  Google Scholar 

  11. Penman S, Smith I, Holtzman E (1966) Ribosomal RNA synthesis and processing in a particulate site in the HeLa cell nucleus. Science 154(750):786–789

    Article  CAS  PubMed  Google Scholar 

  12. Greenberg H, Penman S (1966) Methylation and processing of ribosomal RNA in HeLa cells. J Mol Biol 21(3):527–535

    Article  CAS  PubMed  Google Scholar 

  13. Zimmerman EF, Holler BW (1967) Methylation of 45 s ribosomal RNA precursor in HeLa cells. J Mol Biol 23(2):149–161

    Article  CAS  PubMed  Google Scholar 

  14. Maden BE (1986) Identification of the locations of the methyl groups in 18 S ribosomal RNA from Xenopus laevis and man. J Mol Biol 189(4):681–699

    Article  CAS  PubMed  Google Scholar 

  15. Kos M, Tollervey D (2010) Yeast pre-rRNA processing and modification occur cotranscriptionally. Mol Cell 37(6):809–820. doi:10.1016/j.molcel.2010.02.024, S1097-2765(10)00206-6 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Schwinghamer MW, Shepherd RJ (1980) Formaldehyde-containing slab gels for analysis of denatured, tritium-labeled RNA. Anal Biochem 103(2):426–434

    Article  CAS  PubMed  Google Scholar 

  17. Gurney T Jr (1985) Characterization of mouse 45S ribosomal RNA subspecies suggests that the first processing cleavage occurs 600 +/- 100 nucleotides from the 5′ end and the second 500 +/- 100 nucleotides from the 3′ end of a 13.9 kb precursor. Nucleic Acids Res 13(13):4905–4919

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rosen KM, Lamperti ED, Villa-Komaroff L (1990) Optimizing the northern blot procedure. Biotechniques 8(4):398–403

    CAS  PubMed  Google Scholar 

  19. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

  20. Laskey RA, Mills AD (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56(2):335–341. doi:10.1111/j.1432-1033.1975.tb02238.x

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Tom Moss Ph.D. .

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Stefanovsky, V.Y., Moss, T. (2016). Metabolic Labeling in the Study of Mammalian Ribosomal RNA Synthesis. In: Németh, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_11

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  • DOI: https://doi.org/10.1007/978-1-4939-3792-9_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3790-5

  • Online ISBN: 978-1-4939-3792-9

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