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In Vivo Run-On Assays to Monitor Nascent Precursor RNA Transcripts

  • Piergiorgio PercipalleEmail author
  • Emilie Louvet
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 809)

Abstract

Biochemical methods have provided mechanistic insights into the different transcription phases during which the RNA polymerase is assembled at gene promoter and becomes engaged in the elongation of nascent transcripts. Evidence that transcription takes place in specific regions of the nucleus has fuelled the need to develop assays that can be performed in living cells and provide information on the location of the specific foci, where transcription takes place. In this chapter, we describe a method that is based on the incorporation of a fluorine-conjugated uridine analogue, incorporation that can be monitored by immunofluorescence and light microscopy using specific fluorochrome-conjugated monoclonal antibodies. This assay allows direct monitoring of active transcription foci in living cells. When coupled to suitable software, the method outlined here also provides a semiquantitative approach to measure the number of active transcription foci that correlate with the proliferation state of the cell. Therefore, the assay we present here is a sensitive analytical tool to monitor the topology of transcription foci in the eukaryotic cell nucleus and to gain insight into transcription rates.

Key words

Nascent RNA transcripts Pre-mRNA, transcription inhibitors Immunofluorescence Transcription foci Confocal microscopy Image processing and analysis 

Notes

Acknowledgements

Our work is supported by grants from the Swedish Research Council and Cancerfonden to PP. EL is supported by a postdoctoral fellowship from the Wenner-Gren Foundation, Stockholm, Sweden.

References

  1. 1.
    Alberts B, Johnson A, Lewis J et al (2008) Molecular Biology of the Cell. Garland Science, New YorkGoogle Scholar
  2. 2.
    Eissenberg J C, Shilatifard A (2006) Leaving a mark: the many footprints of the elongating RNA polymerase II. Curr Opin Genet Dev 16:184–190Google Scholar
  3. 3.
    Hirose Y, Ohkuma Y (2007) Phosphorylation of the C-terminal Domain of RNA Polymerase II Plays Central Roles in the Integrated Events of Eucaryotic Gene Expression. J Biochem 141:601–608Google Scholar
  4. 4.
    Rondon A G, Mischo H E, Proudfoot N J (2008) Terminating transcription in yeast: whether to be a ‘nerd’ or a ‘rat’. Nat Struct Mol Biol 15:775–776Google Scholar
  5. 5.
    Jackson D A, Hassan A B, Errington R J et al (1993) Visualization of focal sites of transcription within human nuclei. EMBO J. 12:1059–1065Google Scholar
  6. 6.
    Wansink D G, Schul W, van der Kraan I et al (1993) Fluorescent labelling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol 122:283–293Google Scholar
  7. 7.
    Boisvert F M, van Koningsbruggen S, Navascue´s J et al (2007) The multifunctional nucleolus. Nat Rev Mol Cell Biol 8:574–585Google Scholar
  8. 8.
    Sirri V, Urcuqui-Inchima S, Roussel P et al (2008). Nucleolus: The fascinating nuclear body. Histochem Cell Biol 129:13–31Google Scholar
  9. 9.
    Percipalle P, Fomproix N, Cavellan E et al (2006) The chromatin remodelling complex WSTF-SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription. EMBO Rep 7:525–530Google Scholar
  10. 10.
    Längst G, Blank T A, Becker P B et al (1997) RNA polymerase I transcription on nucleosomal templates: TTF-I induces chromatin remodeling and relieves transcriptional repression. EMBO J 16:760–768Google Scholar
  11. 11.
    Fomproix N, Percipalle P (2004) An actin-myosin complex on actively transcribing genes. Exp Cell Res 294:140–148Google Scholar
  12. 12.
    Moore G P, Ringertz N R (1973) Localization of DNA-dependent RNA polymerase activities in fixed human fibroblasts by autoradiography. Exp Cell Res 76:223–228Google Scholar
  13. 13.
    Mitchell J A, Fraser P (2008) Transcription factories are nuclear subcompartments that remain in the absence of transcription. Genes Dev 22:20–25Google Scholar
  14. 14.
    Kruhlak M, Crouch E E, Orlov M et al (2007) The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks. Nature 447:730–734Google Scholar
  15. 15.
    Percipalle P, Obrdlik A (2009) Analysis of nascent RNA transcripts by chromatin RNA immunoprecipitations. Meth Mol Biol 567:215–235Google Scholar
  16. 16.
    Burger W, Burge M J (2008) Digital Image Processing. Springer, New YorkGoogle Scholar
  17. 17.
    Fraser N W, Sehgal P B, Darnell J E (1978) DRB-induced premature termination of late adenovirus transcription. Nature 272:590–593Google Scholar
  18. 18.
    Marshall N F, Price D H (1995) Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J Biol Chem 270:12335–12338Google Scholar
  19. 19.
    Marshall N F, Peng J, Xie Z et al (1996) Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J Biol Chem 271:27176–27183Google Scholar
  20. 20.
    Gribnau J, Diderich K, Pruzina S et al (2000) Intergenic transcription and developmental remodeling of chromatin subdomains in the human β-globin locus. Mol Cell 5:377–386Google Scholar
  21. 21.
    Louvet E, Tramier M, Angelier N et al (2008) Time-Lapse Microscopy and Fluorescence Resonance Energy Transfer to Analyse the Dynamics and Interactions of Nucleolar Proteins in Living Cells. Meth Mol Biol 463: 123–135Google Scholar
  22. 22.
    Gonzales R C, Woods R E (2008) Digital Image Processing. Pearson Education, LondonGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Cell and Molecular BiologyKarolinska InstitutetStockholmSweden

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