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 comprises 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 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 (IF) (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.
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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 to Steve Artandi, Peter Baumann, Christopher Counter, Kathy Collins, Titia de Lange, Carol Greider, Bill Hahn, Joachim Lingner, Susan Smith, Jerry Shay, Woody Wright, and Songyang (Sunny) Zhou. 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.
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Abreu, E., Terns, R.M., Terns, M.P. (2011). Visualization of Human Telomerase Localization by Fluorescence Microscopy Techniques. In: Songyang, Z. (eds) Telomeres and Telomerase. Methods in Molecular Biology, vol 735. Humana Press. https://doi.org/10.1007/978-1-61779-092-8_12
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DOI: https://doi.org/10.1007/978-1-61779-092-8_12
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