Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro

A Corrigendum to this article was published on 01 May 2006

This article has been updated

Abstract

The ribonucleoprotein enzyme telomerase synthesizes DNA at the ends of chromosomes. Although the telomerase catalytic protein subunit (TERT) is well conserved, the RNA component is rapidly evolving in both size and sequence. Here, we reduce the 1,157-nucleotide (nt) Saccharomyces cerevisiae TLC1 RNA to a size smaller than the 451-nt human RNA while retaining function in vivo. We conclude that long protein-binding arms are not essential for the RNA to serve its scaffolding function. Although viable, cells expressing Mini-T have shortened telomeres and reduced fitness as compared to wild-type cells, suggesting why the larger RNA has evolved. Previous attempts to reconstitute telomerase activity in vitro using TLC1 and yeast TERT (Est2p) have been unsuccessful. We find that substitution of Mini-T for wild-type TLC1 in a reconstituted system yields robust activity, allowing the contributions of individual yeast telomerase components to be directly assessed.

*Note: In the supplementary information initially published online to accompany this article, the secondary structure model for a 436-nt mini-T is shown in Supplementary Figure 1, but the legend describes a 500-nt mini-T model. The secondary structure model for the 500-nt mini-T has now been supplied by the authors. The error has been corrected online.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Design of a smaller yeast telomerase RNA using the secondary structure model for the wild-type RNA.
Figure 2: Reduction of TLC1 to one-third of wild-type (WT) length with retention of function in vivo.
Figure 3: Competitive growth experiments show that the fitness of Mini-T–expressing cells is less than that of wild-type (WT) cells.
Figure 4: Mini-T allows reconstituted yeast telomerase activity in vitro.

Change history

  • 14 April 2006

    File replaced

References

  1. Cech, T.R. Beginning to understand the end of the chromosome. Cell 116, 273–279 (2004).

    CAS  Article  Google Scholar 

  2. Shay, J.W. & Bacchetti, S. A survey of telomerase activity in human cancer. Eur. J. Cancer 33, 787–791 (1997).

    CAS  Article  Google Scholar 

  3. Greider, C.W. & Blackburn, E.H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405–413 (1985).

    CAS  Article  Google Scholar 

  4. Lingner, J. et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276, 561–567 (1997).

    CAS  Article  Google Scholar 

  5. Greider, C.W. & Blackburn, E.H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331–337 (1989).

    CAS  Article  Google Scholar 

  6. Singer, M.S. & Gottschling, D.E. TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266, 404–409 (1994).

    CAS  Article  Google Scholar 

  7. Dandjinou, A.T. et al. A phylogenetically based secondary structure for the yeast telomerase RNA. Curr. Biol. 14, 1148–1158 (2004).

    CAS  Article  Google Scholar 

  8. Zappulla, D.C. & Cech, T.R. Yeast telomerase RNA: a flexible scaffold for protein subunits. Proc. Natl. Acad. Sci. USA 101, 10024–10029 (2004).

    CAS  Article  Google Scholar 

  9. Tzfati, Y., Knight, Z., Roy, J. & Blackburn, E.H. A novel pseudoknot element is essential for the action of a yeast telomerase. Genes Dev. 17, 1779–1788 (2003).

    CAS  Article  Google Scholar 

  10. Livengood, A.J., Zaug, A.J. & Cech, T.R. Essential regions of Saccharomyces cerevisiae telomerase RNA: separate elements for Est1p and Est2p interaction. Mol. Cell. Biol. 22, 2366–2374 (2002).

    CAS  Article  Google Scholar 

  11. Seto, A.G., Livengood, A.J., Tzfati, Y., Blackburn, E. & Cech, T.R. A bulged stem tethers Est1p to telomerase RNA in budding yeasts. Genes Dev. 16, 2800–2810 (2002).

    CAS  Article  Google Scholar 

  12. Peterson, S.E. et al. The function of a stem-loop in telomerase RNA is linked to the DNA repair protein Ku. Nat. Genet. 27, 64–67 (2001).

    CAS  Article  Google Scholar 

  13. Seto, A.G., Zaug, A.J., Sobel, S.G., Wolin, S.L. & Cech, T.R. Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle. Nature 401, 177–180 (1999).

    CAS  Article  Google Scholar 

  14. Ban, N., Nissen, P., Hansen, J., Moore, P.B. & Steitz, T.A. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289, 905–920 (2000).

    CAS  Article  Google Scholar 

  15. Evans, S.K. & Lundblad, V. The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance. Genetics 162, 1101–1115 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Mathews, D.H., Sabina, J., Zuker, M. & Turner, D.H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    CAS  Article  Google Scholar 

  17. Feng, J. et al. The RNA component of human telomerase. Science 269, 1236–1241 (1995).

    CAS  Article  Google Scholar 

  18. Thatcher, J.W., Shaw, J.M. & Dickinson, W.J. Marginal fitness contributions of nonessential genes in yeast. Proc. Natl. Acad. Sci. USA 95, 253–257 (1998).

    CAS  Article  Google Scholar 

  19. Hughes, T.R., Evans, S.K., Weilbaecher, R.G. & Lundblad, V. The Est3 protein is a subunit of yeast telomerase. Curr. Biol. 10, 809–812 (2000).

    CAS  Article  Google Scholar 

  20. Schiestl, R.H. & Gietz, R.D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16, 339–346 (1989).

    CAS  Article  Google Scholar 

  21. Weinrich, S.L. et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat. Genet. 17, 498–502 (1997).

    CAS  Article  Google Scholar 

  22. Beattie, T.L., Zhou, W., Robinson, M.O. & Harrington, L. Reconstitution of human telomerase activity in vitro. Curr. Biol. 8, 177–180 (1998).

    CAS  Article  Google Scholar 

  23. Collins, K. & Gandhi, L. The reverse transcriptase component of the Tetrahymena telomerase ribonucleoprotein complex. Proc. Natl. Acad. Sci. USA 95, 8485–8490 (1998).

    CAS  Article  Google Scholar 

  24. Friedman, K.L., Heit, J.J., Long, D.M. & Cech, T.R. N-terminal domain of yeast telomerase reverse transcriptase: recruitment of Est3p to the telomerase complex. Mol. Biol. Cell 14, 1–13 (2003).

    CAS  Article  Google Scholar 

  25. Cohn, M. & Blackburn, E.H. Telomerase in yeast. Science 269, 396–400 (1995).

    CAS  Article  Google Scholar 

  26. Seto, A.G. et al. A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity. RNA 9, 1323–1332 (2003).

    CAS  Article  Google Scholar 

  27. Morris, D.K. & Lundblad, V. Programmed translational frameshifting in a gene required for yeast telomere replication. Curr. Biol. 7, 969–976 (1997).

    CAS  Article  Google Scholar 

  28. Schroeder, R., Barta, A. & Semrad, K. Strategies for RNA folding and assembly. Nat. Rev. Mol. Cell Biol. 5, 908–919 (2004).

    CAS  Article  Google Scholar 

  29. Uhlenbeck, O.C. Keeping RNA happy. RNA 1, 4–6 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Prescott, J. & Blackburn, E.H. Telomerase RNA mutations in Saccharomyces cerevisiae alter telomerase action and reveal nonprocessivity in vivo and in vitro. Genes Dev. 11, 528–540 (1997).

    CAS  Article  Google Scholar 

  31. Teixeira, M.T., Arneric, M., Sperisen, P. & Lingner, J. Telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states. Cell 117, 323–335 (2004).

    CAS  Article  Google Scholar 

  32. Schmitt, M.E., Brown, T.A. & Trumpower, B.L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 18, 3091–3092 (1990).

    CAS  Article  Google Scholar 

  33. Chapon, C., Cech, T.R. & Zaug, A.J. Polyadenylation of telomerase RNA in budding yeast. RNA 3, 1337–1351 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Lei, M., Zaug, A.J., Podell, E.R. & Cech, T.R. Switching human telomerase on and off with hPOT1 protein in vitro. J. Biol. Chem. 280, 20449–20456 (2005).

    CAS  Article  Google Scholar 

  35. Friedman, K.L. & Cech, T.R. Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants. Genes Dev. 13, 2863–2874 (1999).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A.J. Zaug (University of Colorado, Boulder, Colorado, USA) for plasmid T7-ProA-Est2p and A.G. Seto (present affiliation: Harvard Medical School, Cambridge, Massachusetts, USA) for telomerase immunopurified from yeast. Thanks also to A.J. Zaug for performing the telomerase activity assays that included human TERT. This research was supported in part by grant GM28039 from the US National Institutes of Health.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Thomas R Cech.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Mfold secondary structure model of Mini-T(500). (PDF 907 kb)

Supplementary Fig. 2

Mini-T(500) RNA accumulation when expressed from the chromosome. (PDF 355 kb)

Supplementary Fig. 3

Mini-T RNA levels during log phase cell growth. (PDF 734 kb)

Supplementary Fig. 4

Mini-T(64) and (67) do not yield reconstituted activity. (PDF 586 kb)

Supplementary Fig. 5

Reconstituted core mini-telomerase has activity very similar to wild-type telomerase from yeast. (PDF 1039 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zappulla, D., Goodrich, K. & Cech, T. A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nat Struct Mol Biol 12, 1072–1077 (2005). https://doi.org/10.1038/nsmb1019

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1019

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing