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The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

Abstract

The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.

Abbreviations

CR:

conserved region

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

IP:

immunoprecipitation

ITAS:

internal telomerase assay standard

MDV:

Marek’s disease virus

RNP:

ribonucleoprotein

RRL:

rabbit reticulocyte lysate

RT:

reverse transcription

TERT:

telomerase reverse transcriptase

TR:

telomerase RNA

TRAP:

telomere repeat amplification protocol

References

  1. Palm, W. and de Lange, T. How shelterin protects mammalian telomeres. Annu. Rev. Genet. 42 (2008) 301–334.

    PubMed  Article  CAS  Google Scholar 

  2. Hug, N. and Lingner, J. Telomere length homeostasis. Chromosoma 115 (2006) 413–425.

    PubMed  Article  CAS  Google Scholar 

  3. Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L., Coviello, G.M., Wright, W.E., Weinrich, S.L. and Shay, J.W. Specific association of human telomerase activity with immortal cells and cancer. Science 266 (1994) 2011–2015.

    PubMed  Article  CAS  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  5. Autexier, C. and Lue, N.F. The Structure and Function of Telomerase Reverse Transcriptase. Annu. Rev. Biochem. 75 (2006) 493–517.

    PubMed  Article  CAS  Google Scholar 

  6. Collins, K. The biogenesis and regulation of telomerase holoenzymes. Nat. Rev. Mol. Cell. Biol. 7 (2006) 484–494.

    PubMed  Article  CAS  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  8. Weinrich, S.L., Pruzan, R., Ma, L., Ouellette, M., Tesmer, V.M., Holt, S.E., Bodnar, A.G., Lichtsteiner, S., Kim, N.W., Trager, J.B., Taylor, R.D., Carlos, R., Andrews, W.H., Wright, W.E., Shay, J.W., Harley, C.B. and Morin, G.B. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat. Genet. 17 (1997) 498–502.

    PubMed  Article  CAS  Google Scholar 

  9. Lue, N.F. Adding to the ends: what makes telomerase processive and how important is it? Bioessays 26 (2004) 955–962.

    PubMed  Article  CAS  Google Scholar 

  10. Morin, G.B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59 (1989) 521–529.

    PubMed  Article  CAS  Google Scholar 

  11. Prowse, K.R., Avilion, A.A. and Greider, C.W. Identification of a nonprocessive telomerase activity from mouse cells. Proc. Natl. Acad. Sci. USA 90 (1993) 1493–1497.

    PubMed  Article  CAS  Google Scholar 

  12. Ly, H., Blackburn, E.H. and Parslow, T.G. Comprehensive structurefunction analysis of the core domain of human telomerase RNA. Mol. Cell. Biol. 23 (2003) 6849–6856.

    PubMed  Article  CAS  Google Scholar 

  13. Chen, J.L. and Greider, C.W. Determinants in mammalian telomerase RNA that mediate enzyme processivity and cross-species incompatibility. EMBO J. 22 (2003) 304–314.

    PubMed  Article  Google Scholar 

  14. Moriarty, T.J., Marie-Egyptienne, D.T. and Autexier, C. Functional organization of repeat addition processivity and DNA synthesis determinants in the human telomerase multimer. Mol. Cell. Biol. 24 (2004) 3720–3733.

    PubMed  Article  CAS  Google Scholar 

  15. Moriarty, T.J., Ward, R.J., Taboski, M.A. and Autexier, C. An anchor sitetype defect in human telomerase that disrupts telomere length maintenance and cellular immortalization. Mol. Biol. Cell. 16 (2005) 3152–3161.

    PubMed  Article  CAS  Google Scholar 

  16. Rivera, M.A. and Blackburn, E.H. Processive utilization of the human telomerase template: lack of a requirement for template switching. J. Biol. Chem. 279 (2004) 53770–53781.

    PubMed  Article  CAS  Google Scholar 

  17. Huard, S., Moriarty, T.J. and Autexier, C. The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity. Nucleic Acids Res. 31 (2003) 4059–4070.

    PubMed  Article  CAS  Google Scholar 

  18. Barsov, E.V. Immortalization of human and rhesus macaque primary antigen-specific T cells by retrovirally transduced telomerase reverse transcriptase. Curr. Protoc. Immunol. Chapter 7 (2011) Unit7 21B.

  19. Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S. and Wright, W.E. Extension of life-span by introduction of telomerase into normal human cells. Science 279 (1998) 349–352.

    PubMed  Article  CAS  Google Scholar 

  20. Sagong, M., Park, C.K., Kim, S.H., Lee, K.K., Lee, O.S., Lee, D.S., Cha, S.Y. and Lee, C. Human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus. J. Virol. Methods 179 (2011) 26–32.

    PubMed  Article  Google Scholar 

  21. Veitonmäki, N., Fuxe, J., Hultdin, M., Roos, G., Pettersson, R.F. and Cao, Y. Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4A/pRb. FASEB J. 17 (2003) 764–766.

    PubMed  Google Scholar 

  22. Xiang, H., Wang, J., Mao, Y.W. and Li, D.W. hTERT can function with rabbit telomerase RNA: regulation of gene expression and attenuation of apoptosis. Biochem. Biophys. Res. Commun. 278 (2000) 503–510.

    PubMed  Article  CAS  Google Scholar 

  23. Zou, Y., Yi, X., Wright, W.E. and Shay, J.W. Human telomerase can immortalize Indian muntjac cells. Exp. Cell. Res. 281 (2002) 63–76.

    PubMed  Article  CAS  Google Scholar 

  24. Wang, J., Feng, H., Huang, X.Q., Xiang, H., Mao, Y.W., Liu, J.P., Yan, Q., Liu, W.B., Liu, Y., Deng, M., Gong, L., Sun, S., Luo, C., Liu, S.J., Zhang, X.J. and Li, D.W. Human telomerase reverse transcriptase immortalizes bovine lens epithelial cells and suppresses differentiation through regulation of the ERK signaling pathway. J. Biol. Chem. 280 (2005) 22776–22787.

    PubMed  Article  CAS  Google Scholar 

  25. Fakhoury, J., Marie-Egyptienne, D.T., Londono-Vallejo, J.A. and Autexier, C. Telomeric function of mammalian telomerases at short telomeres. J. Cell. Sci. 123 (2010) 1693–1704.

    PubMed  Article  CAS  Google Scholar 

  26. Middleman, E.J., Choi, J., Venteicher, A.S., Cheung, P. and Artandi, S.E. Regulation of cellular immortalization and steady-state levels of the telomerase reverse transcriptase through its carboxy-terminal domain. Mol. Cell. Biol. 26 (2006) 2146–2159.

    PubMed  Article  CAS  Google Scholar 

  27. Wong, S.C., Ong, L.L., Er, C.P., Gao, S., Yu, H. and So, J.B. Cloning of rat telomerase catalytic subunit functional domains, reconstitution of telomerase activity and enzymatic profile of pig and chicken tissues. Life Sci. 73 (2003) 2749–2760.

    PubMed  Article  CAS  Google Scholar 

  28. Fragnet, L., Blasco, M.A., Klapper, W. and Rasschaert, D. The RNA subunit of telomerase is encoded by Marek’s disease virus. J. Virol. 77 (2003) 5985–5996.

    PubMed  Article  CAS  Google Scholar 

  29. Osterrieder, N., Kamil, J.P., Schumacher, D., Tischer, B.K. and Trapp, S. Marek’s disease virus: from miasma to model. Nat. Rev. Microbiol. 4 (2006) 283–294.

    PubMed  Article  CAS  Google Scholar 

  30. Trapp, S., Parcells, M.S., Kamil, J.P., Schumacher, D., Tischer, B.K., Kumar, P.M., Nair, V.K. and Osterrieder, N. A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis. J. Exp. Med. 203 (2006) 1307–1317.

    PubMed  Article  CAS  Google Scholar 

  31. Kaufer, B.B., Arndt, S., Trapp, S., Osterrieder, N. and Jarosinski, K.W. Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis. PLoS Pathog. 7 (2011) e1002333.

    PubMed  Article  CAS  Google Scholar 

  32. Shkreli, M., Dambrine, G., Soubieux, D., Kut, E. and Rasschaert, D. Involvement of the Oncoprotein c-Myc in Viral Telomerase RNA Gene Regulation during Marek’s Disease Virus-Induced Lymphomagenesis. J. Virol. 81 (2007) 4848–4857.

    PubMed  Article  CAS  Google Scholar 

  33. Fragnet, L., Kut, E. and Rasschaert, D. Comparative functional study of the viral telomerase RNA based on natural mutations. J. Biol. Chem. 280 (2005) 23502–23515.

    PubMed  Article  CAS  Google Scholar 

  34. Chen, J.L., Opperman, K.K. and Greider, C.W. A critical stem-loop structure in the CR4-CR5 domain of mammalian telomerase RNA. Nucleic Acids Res. 30 (2002) 592–597.

    PubMed  Article  CAS  Google Scholar 

  35. Chen, J.L., Blasco, M.A. and Greider, C.W. Secondary structure of vertebrate telomerase RNA. Cell 100 (2000) 503–514.

    PubMed  Article  CAS  Google Scholar 

  36. Autexier, C., Pruzan, R., Funk, W.D. and Greider, C.W. Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA. EMBO J. 15 (1996) 5928–5935.

    PubMed  CAS  Google Scholar 

  37. Beattie, T.L., Zhou, W., Robinson, M.O. and Harrington, L. Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding. Mol. Biol. Cell. 11 (2000) 3329–3340.

    PubMed  CAS  Google Scholar 

  38. Guiducci, C., Cerone, M.A. and Bacchetti, S. Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability. Oncogene 20 (2001) 714–725.

    PubMed  Article  CAS  Google Scholar 

  39. Nakamura, T.M., Morin, G.B., Chapman, K.B., Weinrich, S.L., Andrews, W.H., Lingner, J., Harley, C.B. and Cech, T.R. Telomerase catalytic subunit homologs from fission yeast and human. Science 277 (1997) 955–959.

    PubMed  Article  CAS  Google Scholar 

  40. Marie-Egyptienne, D.T., Cerone, M.A., Londono-Vallejo, J.A. and Autexier, C. A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation. Nucleic Acids Res. 33 (2005) 5446–5457.

    PubMed  Article  CAS  Google Scholar 

  41. Mitchell, J.R. and Collins, K. Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase. Mol. Cell. 6 (2000) 361–371.

    PubMed  Article  CAS  Google Scholar 

  42. Bachand, F. and Autexier, C. Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions. Mol. Cell. Biol. 21 (2001) 1888–1897.

    PubMed  Article  CAS  Google Scholar 

  43. Lai, C.K., Mitchell, J.R. and Collins, K. RNA binding domain of telomerase reverse transcriptase. Mol. Cell. Biol. 21 (2001) 990–1000.

    PubMed  Article  CAS  Google Scholar 

  44. Leeper, T., Leulliot, N. and Varani, G. The solution structure of an essential stem-loop of human telomerase RNA. Nucleic Acids Res. 31 (2003) 2614–2621.

    PubMed  Article  CAS  Google Scholar 

  45. Ueda, C.T. and Roberts, R.W. Analysis of a long-range interaction between conserved domains of human telomerase RNA. RNA 10 (2004) 139–147.

    PubMed  Article  CAS  Google Scholar 

  46. Cerone, M.A., Londono-Vallejo, J.A. and Bacchetti, S. Telomere maintenance by telomerase and by recombination can coexist in human cells. Hum. Mol. Genet. 10 (2001) 1945–1952.

    PubMed  Article  CAS  Google Scholar 

  47. Cerone, M.A., Ward, R.J., Londono-Vallejo, J.A. and Autexier, C. Telomerase RNA mutated in autosomal dyskeratosis congenita reconstitutes a weakly active telomerase enzyme defective in telomere elongation. Cell Cycle 4 (2005) 585–589.

    PubMed  Article  CAS  Google Scholar 

  48. Fu, D. and Collins, K. Distinct biogenesis pathways for human telomerase RNA and H/ACA small nucleolar RNAs. Mol. Cell. 11 (2003) 1361–1372.

    PubMed  Article  CAS  Google Scholar 

  49. Tesmer, V.M., Ford, L.P., Holt, S.E., Frank, B.C., Yi, X., Aisner, D.L., Ouellette, M., Shay, J.W. and Wright, W.E. Two inactive fragments of the integral RNA cooperate to assemble active telomerase with the human protein catalytic subunit (hTERT) in vitro. Mol. Cell. Biol. 19 (1999) 6207–6216.

    PubMed  CAS  Google Scholar 

  50. Wen, J., Cong, Y.S. and Bacchetti, S. Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells. Hum. Mol. Genet. 7 (1998) 1137–1141.

    PubMed  Article  CAS  Google Scholar 

  51. Cong, Y.S., Wright, W.E. and Shay, J.W. Human telomerase and its regulation. Microbiol. Mol. Biol. Rev. 66 (2002) 407–425.

    PubMed  Article  CAS  Google Scholar 

  52. Harrington, L. Biochemical aspects of telomerase function. Cancer Lett. 194 (2003) 139–154.

    PubMed  Article  CAS  Google Scholar 

  53. Dez, C., Henras, A., Faucon, B., Lafontaine, D., Caizergues-Ferrer, M. and Henry, Y. Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p. Nucleic Acids Res. 29 (2001) 598–603.

    PubMed  Article  CAS  Google Scholar 

  54. Dragon, F., Pogacic, V. and Filipowicz, W. In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs. Mol. Cell. Biol. 20 (2000) 3037–3048.

    PubMed  Article  CAS  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  56. Mitchell, J.R., Wood, E. and Collins, K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402 (1999) 551–555.

    PubMed  Article  CAS  Google Scholar 

  57. Seimiya, H., Sawada, H., Muramatsu, Y., Shimizu, M., Ohko, K., Yamane, K. and Tsuruo, T. Involvement of 14-3-3 proteins in nuclear localization of telomerase. EMBO J. 19 (2000) 2652–2661.

    PubMed  Article  CAS  Google Scholar 

  58. Cohen, S.B., Graham, M.E., Lovrecz, G.O., Bache, N., Robinson, P.J. and Reddel, R.R. Protein composition of catalytically active human telomerase from immortal cells. Science 315 (2007) 1850–1853.

    PubMed  Article  CAS  Google Scholar 

  59. Marrone, A. and Mason, P.J. Dyskeratosis congenita. Cell. Mol. Life Sci. 60 (2003) 507–517.

    PubMed  Article  CAS  Google Scholar 

  60. Mitchell, J.R., Cheng, J. and Collins, K. A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3′ end. Mol. Cell. Biol. 19 (1999) 567–576.

    PubMed  CAS  Google Scholar 

  61. Wong, J.M. and Collins, K. Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita. Genes Dev. 20 (2006) 2848–2858.

    PubMed  Article  CAS  Google Scholar 

  62. Michailidis, G., Saretzki, G. and Hall, J. Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts. Biochem. Biophys. Res. Commun. 335 (2005) 240–246.

    PubMed  Article  CAS  Google Scholar 

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Correspondence to Laetitia Trapp-Fragnet.

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Trapp-Fragnet, L., Marie-Egyptienne, D.T., Fakhoury, J. et al. The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells. Cell Mol Biol Lett 17, 598–615 (2012). https://doi.org/10.2478/s11658-012-0031-6

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Key words

  • Telomerase
  • Holoenzyme
  • hTERT
  • vTR
  • Dyskerin
  • Marek’s disease virus