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. 1996 Nov 1;15(21):5928–5935.

Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA.

C Autexier 1, R Pruzan 1, W D Funk 1, C W Greider 1
PMCID: PMC452365  PMID: 8918470

Abstract

Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5' and 3' ends of hTR identified a functional region of hTR, similar in size to the full-length telomerase RNAs from ciliates. This region is located between positions 1-203. Furthermore, we found that residues 1-44, 5' to the template region (residues 46-56) are not essential for activity, indicating a minimal functional region is located between residues 44-203. Mutagenesis of full-length hTR between residues 170-179, 180-189 or 190-199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allsopp R. C., Vaziri H., Patterson C., Goldstein S., Younglai E. V., Futcher A. B., Greider C. W., Harley C. B. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10114–10118. doi: 10.1073/pnas.89.21.10114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ares M., Jr U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs. Cell. 1986 Oct 10;47(1):49–59. doi: 10.1016/0092-8674(86)90365-x. [DOI] [PubMed] [Google Scholar]
  3. Autexier C., Greider C. W. Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. Genes Dev. 1995 Sep 15;9(18):2227–2239. doi: 10.1101/gad.9.18.2227. [DOI] [PubMed] [Google Scholar]
  4. Autexier C., Greider C. W. Functional reconstitution of wild-type and mutant Tetrahymena telomerase. Genes Dev. 1994 Mar 1;8(5):563–575. doi: 10.1101/gad.8.5.563. [DOI] [PubMed] [Google Scholar]
  5. Blasco M. A., Funk W., Villeponteau B., Greider C. W. Functional characterization and developmental regulation of mouse telomerase RNA. Science. 1995 Sep 1;269(5228):1267–1270. doi: 10.1126/science.7544492. [DOI] [PubMed] [Google Scholar]
  6. Collins K., Kobayashi R., Greider C. W. Purification of Tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme. Cell. 1995 Jun 2;81(5):677–686. doi: 10.1016/0092-8674(95)90529-4. [DOI] [PubMed] [Google Scholar]
  7. Counter C. M., Avilion A. A., LeFeuvre C. E., Stewart N. G., Greider C. W., Harley C. B., Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 1992 May;11(5):1921–1929. doi: 10.1002/j.1460-2075.1992.tb05245.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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. The RNA component of human telomerase. Science. 1995 Sep 1;269(5228):1236–1241. doi: 10.1126/science.7544491. [DOI] [PubMed] [Google Scholar]
  9. Greider C. W., Blackburn E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989 Jan 26;337(6205):331–337. doi: 10.1038/337331a0. [DOI] [PubMed] [Google Scholar]
  10. Greider C. W., Blackburn E. H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985 Dec;43(2 Pt 1):405–413. doi: 10.1016/0092-8674(85)90170-9. [DOI] [PubMed] [Google Scholar]
  11. Greider C. W. Telomere length regulation. Annu Rev Biochem. 1996;65:337–365. doi: 10.1146/annurev.bi.65.070196.002005. [DOI] [PubMed] [Google Scholar]
  12. Harley C. B., Futcher A. B., Greider C. W. Telomeres shorten during ageing of human fibroblasts. Nature. 1990 May 31;345(6274):458–460. doi: 10.1038/345458a0. [DOI] [PubMed] [Google Scholar]
  13. Harley C. B., Kim N. W., Prowse K. R., Weinrich S. L., Hirsch K. S., West M. D., Bacchetti S., Hirte H. W., Counter C. M., Greider C. W. Telomerase, cell immortality, and cancer. Cold Spring Harb Symp Quant Biol. 1994;59:307–315. doi: 10.1101/sqb.1994.059.01.035. [DOI] [PubMed] [Google Scholar]
  14. Hastie N. D., Dempster M., Dunlop M. G., Thompson A. M., Green D. K., Allshire R. C. Telomere reduction in human colorectal carcinoma and with ageing. Nature. 1990 Aug 30;346(6287):866–868. doi: 10.1038/346866a0. [DOI] [PubMed] [Google Scholar]
  15. Igel A. H., Ares M., Jr Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing. Nature. 1988 Aug 4;334(6181):450–453. doi: 10.1038/334450a0. [DOI] [PubMed] [Google Scholar]
  16. 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., Shay J. W. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994 Dec 23;266(5193):2011–2015. doi: 10.1126/science.7605428. [DOI] [PubMed] [Google Scholar]
  17. Lindsey J., McGill N. I., Lindsey L. A., Green D. K., Cooke H. J. In vivo loss of telomeric repeats with age in humans. Mutat Res. 1991 Jan;256(1):45–48. doi: 10.1016/0921-8734(91)90032-7. [DOI] [PubMed] [Google Scholar]
  18. Lingner J., Hendrick L. L., Cech T. R. Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev. 1994 Aug 15;8(16):1984–1998. doi: 10.1101/gad.8.16.1984. [DOI] [PubMed] [Google Scholar]
  19. Lumelsky N., Altman S. Selection and characterization of randomly produced mutants in the gene coding for M1 RNA. J Mol Biol. 1988 Aug 5;202(3):443–454. doi: 10.1016/0022-2836(88)90277-x. [DOI] [PubMed] [Google Scholar]
  20. McCormick-Graham M., Romero D. P. A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium. Mol Cell Biol. 1996 Apr;16(4):1871–1879. doi: 10.1128/mcb.16.4.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McEachern M. J., Blackburn E. H. Runaway telomere elongation caused by telomerase RNA gene mutations. Nature. 1995 Aug 3;376(6539):403–409. doi: 10.1038/376403a0. [DOI] [PubMed] [Google Scholar]
  22. Nierhaus K. H. The assembly of prokaryotic ribosomes. Biochimie. 1991 Jun;73(6):739–755. doi: 10.1016/0300-9084(91)90054-5. [DOI] [PubMed] [Google Scholar]
  23. Olovnikov A. M. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol. 1973 Sep 14;41(1):181–190. doi: 10.1016/0022-5193(73)90198-7. [DOI] [PubMed] [Google Scholar]
  24. Romero D. P., Blackburn E. H. A conserved secondary structure for telomerase RNA. Cell. 1991 Oct 18;67(2):343–353. doi: 10.1016/0092-8674(91)90186-3. [DOI] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shumyatsky G., Reddy R. Compilation of small RNA sequences. Nucleic Acids Res. 1992 May 11;20 (Suppl):2159–2165. doi: 10.1093/nar/20.suppl.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shuster E. O., Guthrie C. Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides. Cell. 1988 Oct 7;55(1):41–48. doi: 10.1016/0092-8674(88)90007-4. [DOI] [PubMed] [Google Scholar]
  28. Siliciano P. G., Kivens W. J., Guthrie C. More than half of yeast U1 snRNA is dispensable for growth. Nucleic Acids Res. 1991 Dec 11;19(23):6367–6372. doi: 10.1093/nar/19.23.6367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Singer M. S., Gottschling D. E. TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science. 1994 Oct 21;266(5184):404–409. doi: 10.1126/science.7545955. [DOI] [PubMed] [Google Scholar]
  30. Strub K., Moss J., Walter P. Binding sites of the 9- and 14-kilodalton heterodimeric protein subunit of the signal recognition particle (SRP) are contained exclusively in the Alu domain of SRP RNA and contain a sequence motif that is conserved in evolution. Mol Cell Biol. 1991 Aug;11(8):3949–3959. doi: 10.1128/mcb.11.8.3949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ségault V., Will C. L., Sproat B. S., Lührmann R. In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs. EMBO J. 1995 Aug 15;14(16):4010–4021. doi: 10.1002/j.1460-2075.1995.tb00072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vaziri H., Schächter F., Uchida I., Wei L., Zhu X., Effros R., Cohen D., Harley C. B. Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet. 1993 Apr;52(4):661–667. [PMC free article] [PubMed] [Google Scholar]
  33. Waugh D. S., Green C. J., Pace N. R. The design and catalytic properties of a simplified ribonuclease P RNA. Science. 1989 Jun 30;244(4912):1569–1571. doi: 10.1126/science.2472671. [DOI] [PubMed] [Google Scholar]
  34. Wright W. E., Shay J. W. Time, telomeres and tumours: is cellular senescence more than an anticancer mechanism? Trends Cell Biol. 1995 Aug;5(8):293–297. doi: 10.1016/s0962-8924(00)89044-3. [DOI] [PubMed] [Google Scholar]
  35. Yu G. L., Bradley J. D., Attardi L. D., Blackburn E. H. In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature. 1990 Mar 8;344(6262):126–132. doi: 10.1038/344126a0. [DOI] [PubMed] [Google Scholar]
  36. Zaug A. J., Linger J., Cech T. R. Method for determining RNA 3' ends and application to human telomerase RNA. Nucleic Acids Res. 1996 Feb 1;24(3):532–533. doi: 10.1093/nar/24.3.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. de Lange T., Shiue L., Myers R. M., Cox D. R., Naylor S. L., Killery A. M., Varmus H. E. Structure and variability of human chromosome ends. Mol Cell Biol. 1990 Feb;10(2):518–527. doi: 10.1128/mcb.10.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]

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