Skip to main content
SpringerLink
Log in
Book cover

Telomeres and Telomerase pp 113–125Cite as

Visualization of Human Telomerase Localization by Fluorescence Microscopy Techniques

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1587))

Abstract

Human telomerase is a ribonucleoprotein (RNP) that synthesizes DNA repeats at the ends of chromosomes and maintains telomere length and genome stability. The enzyme is comprised of telomerase RNA (hTR) (which provides the template for telomere addition) and several protein subunits including telomerase reverse transcriptase (hTERT) (the catalytic component). Intracellular trafficking of the enzyme has emerged as an important factor in the regulation of telomerase activity. Telomerase trafficking between nuclear Cajal bodies (proposed sites of telomerase biogenesis and regulation) and telomeres (sites of action) is regulated by the cell cycle in concordance with telomere synthesis during S phase. Here, we describe fluorescence microscopy approaches to visualize the subcellular localization of the essential RNA component of telomerase (hTR) relative to Cajal bodies and telomeres in cultured human cells. These methods include fluorescence in situ hybridization (to detect hTR and telomeric DNA) and immunofluorescence (to detect Cajal bodies and telomere binding proteins). Because telomerase localization to telomeres is normally restricted to S phase, we also describe methods to synchronize and analyze cells within this phase of the cell cycle.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Autexier C, Lue NF (2006) The structure and function of telomerase reverse transcriptase. Annu Rev Biochem 75:493–517

    Article  CAS  PubMed  Google Scholar 

  2. Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, Bodnar AG, Lichtsteiner S, Kim NW, Trager JB, Taylor RD, Carlos R, Andrews WH, Wright WE, Shay JW, Harley CB, Morin GB (1997) Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 17:498–502

    Article  CAS  PubMed  Google Scholar 

  3. Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ, Reddel RR (2007) Protein composition of catalytically active human telomerase from immortal cells. Science 315:1850–1853

    Article  CAS  PubMed  Google Scholar 

  4. Fu D, Collins K (2007) Purification of human telomerase complexes identifies factors involved in telomerase biogenesis and telomere length regulation. Mol Cell 28:773–785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pogacic V, Dragon F, Filipowicz W (2000) Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10. Mol Cell Biol 20:9028–9040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Reichenbach P, Hoss M, Azzalin CM, Nabholz M, Bucher P, Lingner J (2003) A human homolog of yeast Est1 associates with telomerase and uncaps chromosome ends when overexpressed. Curr Biol 13:568–574

    Article  CAS  PubMed  Google Scholar 

  7. Snow BE, Erdmann N, Cruickshank J, Goldman H, Gill RM, Robinson MO, Harrington L (2003) Functional conservation of the telomerase protein Est1p in humans. Curr Biol 13:698–704

    Article  CAS  PubMed  Google Scholar 

  8. Tycowski KT, Shu MD, Kukoyi A, Steitz JA (2009) A conserved WD40 protein binds the cajal body localization signal of scaRNP particles. Mol Cell 34:47–57

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Venteicher AS, Abreu EB, Meng Z, McCann KE, Terns RM, Veenstra TD, Terns MP, Artandi SE (2009) A human telomerase holoenzyme protein required for cajal body localization and telomere synthesis. Science 323:644–648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Collins K, Mitchell JR (2002) Telomerase in the human organism. Oncogene 21:564–579

    Article  CAS  PubMed  Google Scholar 

  11. Cong YS, Wright WE, Shay JW (2002) Human telomerase and its regulation. Microbiol Mol Biol Rev 66:407–425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Feng, J., Funk, W. D., Wang, S. S., Weinrich, S. L., Avilion, A. A., Chiu, C. P., Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., and al, e. (1995) The RNA component of human telomerase, Science 269, 1236–1241.

    Google Scholar 

  13. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352

    Article  CAS  PubMed  Google Scholar 

  14. Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA (1999) Creation of human tumour cells with defined genetic elements. Nature 400:464–468

    Article  CAS  PubMed  Google Scholar 

  15. Cristofari G, Adolf E, Reichenbach P, Sikora K, Terns RM, Terns MP, Lingner J (2007) Human telomerase RNA accumulation in cajal bodies facilitates telomerase recruitment to telomeres and telomere elongation. Mol Cell 27:882–889

    Article  CAS  PubMed  Google Scholar 

  16. Jady BE, Bertrand E, Kiss T (2004) Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal. J Cell Biol 164:647–652

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jady BE, Richard P, Bertrand E, Kiss T (2006) Cell cycle-dependent recruitment of telomerase RNA and cajal bodies to human telomeres. Mol Biol Cell 17:944–954

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tomlinson RL, Abreu EB, Ziegler T, Ly H, Counter CM, Terns RM, Terns MP (2008) Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells. Mol Biol Cell 19:3793–3800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tomlinson RL, Ziegler TD, Supakorndej T, Terns RM, Terns MP (2006) Cell cycle-regulated trafficking of human telomerase to telomeres. Mol Biol Cell 17:955–965

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhu Y, Tomlinson RL, Lukowiak AA, Terns RM, Terns MP (2004) Telomerase RNA accumulates in Cajal bodies in human cancer cells. Mol Biol Cell 15:81–90

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cioce M, Lamond AI (2005) Cajal bodies: a long history of discovery. Annu Rev Cell Dev Biol 21:105–131

    Article  CAS  PubMed  Google Scholar 

  22. Gall JG (2003) The centennial of the Cajal body. Nat Rev Mol Cell Biol 4:975–980

    Article  CAS  PubMed  Google Scholar 

  23. Cristofari G, Lingner J (2006) Telomere length homeostasis requires that telomerase levels are limiting. EMBO J 25:565–574

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. O’Keefe RT, Henderson SC, Spector DL (1992) Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences. J Cell Biol 116:1095–1110

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank current and former members of the Terns lab who helped establish and refine the techniques described in this chapter with special acknowledgment to Andrew Lukowiak, Yusheng Zhu, and Rebecca Tomlinson, for their early role in establishing the methodology described here. We are also very grateful to the numerous members of the telomere/telomerase community for their generosity in providing cell lines, antibodies, and constructs critical for this work. In this regard, special thanks are given to Steve Artandi, Peter Baumann, Christopher Counter, Kathy Collins, Titia de Lange, Carol Greider, Bill Hahn, Joachim Lingner, Jerry Shay, Woody Wright, and Zhou (Sunny) Songyang. This work was supported by a National Cancer Institute (NCI) grant (R01 CA104676) to MPT and RMT and a National Institutes of Health Ruth L. Kirschstein NRSA Predoctoral fellowship to EA.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Georgia, A326B Life Sciences Bldg, Athens, GA, 30602, USA

    Eladio Abreu, Rebecca M. Terns & Michael P. Terns

  2. Department of Genetics, University of Georgia, Athens, GA, 30602, USA

    Eladio Abreu, Rebecca M. Terns & Michael P. Terns

Authors
  1. Eladio Abreu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebecca M. Terns
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael P. Terns
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael P. Terns .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA

    Zhou Songyang

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Abreu, E., Terns, R.M., Terns, M.P. (2017). Visualization of Human Telomerase Localization by Fluorescence Microscopy Techniques. In: Songyang, Z. (eds) Telomeres and Telomerase. Methods in Molecular Biology, vol 1587. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6892-3_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6892-3_11

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6891-6

  • Online ISBN: 978-1-4939-6892-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation