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.

  • Article
  • Published:

Structural basis for protein-RNA recognition in telomerase

Nature Structural & Molecular Biology volume 21, pages 507–512 (2014)Cite this article

Subjects

Abstract

Telomerase is a large ribonucleoprotein complex minimally composed of a catalytic telomerase reverse transcriptase (TERT) and an RNA component (TR) that provides the template for telomeric DNA synthesis. However, it remains unclear how TERT and TR assemble into a functional telomerase. Here we report the crystal structure of the conserved regions 4 and 5 (CR4/5) of TR in complex with the TR-binding domain (TRBD) of TERT from the teleost fish Oryzias latipes. The structure shows that CR4/5 adopts an L-shaped three-way-junction conformation with its two arms clamping onto TRBD. Both the sequence and conformation of CR4/5 are required for the interaction. Our structural and mutational analyses suggest that the observed CR4/5-TRBD recognition is common to most eukaryotes, and CR4/5 in vertebrate TR might have a similar role in telomerase regulation as that of stem-loop IV in Tetrahymena TR.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of the CR4/5–TRBD complex structure.
Figure 2: Comparison of CR4/5 conformations in free form and in the CR4/5–TRBD complex.
Figure 3: Structural and mutational analysis of the CR4/5-TRBD interaction.
Figure 4: Phylogenetic and functional analysis of the invariant junction J6-6.1 nucleotide A.
Figure 5: Homology model of the CR4/5–TERT complex.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

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

    Article  CAS  Google Scholar 

  2. Blasco, M.A. Telomeres and human disease: ageing, cancer and beyond. Nat. Rev. Genet. 6, 611–622 (2005).

    Article  CAS  Google Scholar 

  3. Shay, J.W. & Wright, W.E. Telomerase therapeutics for cancer: challenges and new directions. Nat. Rev. Drug Discov. 5, 577–584 (2006).

    Article  CAS  Google Scholar 

  4. Harley, C.B. Telomerase and cancer therapeutics. Nat. Rev. Cancer 8, 167–179 (2008).

    Article  CAS  Google Scholar 

  5. Autexier, C. & Lue, N.F. The structure and function of telomerase reverse transcriptase. Annu. Rev. Biochem. 75, 493–517 (2006).

    Article  CAS  Google Scholar 

  6. Gillis, A.J., Schuller, A.P. & Skordalakes, E. Structure of the Tribolium castaneum telomerase catalytic subunit TERT. Nature 455, 633–637 (2008).

    Article  CAS  Google Scholar 

  7. Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H. & Skordalakes, E. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat. Struct. Mol. Biol. 17, 513–518 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Chen, J.L. & Greider, C.W. Telomerase RNA structure and function: implications for dyskeratosis congenita. Trends Biochem. Sci. 29, 183–192 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Tesmer, V.M. et al. 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, 6207–6216 (1999).

    Article  CAS  Google Scholar 

  13. Vulliamy, T. et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 413, 432–435 (2001).

    Article  CAS  Google Scholar 

  14. Vulliamy, T., Marrone, A., Dokal, I. & Mason, P.J. Association between aplastic anaemia and mutations in telomerase RNA. Lancet 359, 2168–2170 (2002).

    Article  CAS  Google Scholar 

  15. Robart, A.R. & Collins, K. Human telomerase domain interactions capture DNA for TEN domain-dependent processive elongation. Mol. Cell 42, 308–318 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Brown, Y. et al. A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs. Nucleic Acids Res. 35, 6280–6289 (2007).

    Article  CAS  Google Scholar 

  18. Mason, D.X., Goneska, E. & Greider, C.W. Stem-loop IV of tetrahymena telomerase RNA stimulates processivity in trans. Mol. Cell. Biol. 23, 5606–5613 (2003).

    Article  CAS  Google Scholar 

  19. Richards, R.J. et al. Structural study of elements of Tetrahymena telomerase RNA stem-loop IV domain important for function. RNA 12, 1475–1485 (2006).

    Article  CAS  Google Scholar 

  20. Bley, C.J. et al. RNA-protein binding interface in the telomerase ribonucleoprotein. Proc. Natl. Acad. Sci. USA 108, 20333–20338 (2011).

    Article  CAS  Google Scholar 

  21. Harkisheimer, M., Mason, M., Shuvaeva, E. & Skordalakes, E. A motif in the vertebrate telomerase N-terminal linker of TERT contributes to RNA binding and telomerase activity and processivity. Structure 21, 1870–1878 (2013).

    Article  CAS  Google Scholar 

  22. Kim, N.K., Theimer, C.A., Mitchell, J.R., Collins, K. & Feigon, J. Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA. Nucleic Acids Res. 38, 6746–6756 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Kim, N.K., Zhang, Q. & Feigon, J. Structure and sequence elements of the CR4/5 domain of medaka telomerase RNA important for telomerase function. Nucleic Acids Res. 42, 3395–3408 (2013).

    Article  Google Scholar 

  25. Xie, M. et al. Structure and function of the smallest vertebrate telomerase RNA from teleost fish. J. Biol. Chem. 283, 2049–2059 (2008).

    Article  CAS  Google Scholar 

  26. Podlevsky, J.D., Bley, C.J., Omana, R.V., Qi, X. & Chen, J.J. The telomerase database. Nucleic Acids Res. 36, D339–D343 (2008).

    Article  CAS  Google Scholar 

  27. Jiang, J. et al. The architecture of Tetrahymena telomerase holoenzyme. Nature 496, 187–192 (2013).

    Article  CAS  Google Scholar 

  28. Zhang, Q., Kim, N.K., Peterson, R.D., Wang, Z. & Feigon, J. Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA. Proc. Natl. Acad. Sci. USA 107, 18761–18768 (2010).

    Article  CAS  Google Scholar 

  29. Cash, D.D. et al. Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo. Proc. Natl. Acad. Sci. USA 110, 10970–10975 (2013).

    Article  CAS  Google Scholar 

  30. Theimer, C.A., Blois, C.A. & Feigon, J. Structure of the human telomerase RNA pseudoknot reveals conserved tertiary interactions essential for function. Mol. Cell 17, 671–682 (2005).

    Article  CAS  Google Scholar 

  31. Qiao, F. & Cech, T.R. Triple-helix structure in telomerase RNA contributes to catalysis. Nat. Struct. Mol. Biol. 15, 634–640 (2008).

    Article  CAS  Google Scholar 

  32. Qiao, F., Goodrich, K.J. & Cech, T.R. Engineering cis-telomerase RNAs that add telomeric repeats to themselves. Proc. Natl. Acad. Sci. USA 107, 4914–4918 (2010).

    Article  CAS  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  34. De La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Article  CAS  Google Scholar 

  35. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  36. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  37. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  38. Qi, X. et al. The common ancestral core of vertebrate and fungal telomerase RNAs. Nucleic Acids Res. 41, 450–462 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Y. Li (University of Michigan) and K. Wan (University of Michigan) for technical support and F. Guo (University of California, Los Angeles) for suggestions. M.L. is supported as a Howard Hughes Medical Institute Early Career Scientist. This work was supported by grants from the Ministry of Science and Technology of China (2013CB910400 to M.L.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08010201 to M.L.) and the US National Institutes of Health (RO1GM094450 to J.J.-L.C.).

Author information

Authors and Affiliations

  1. National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Jing Huang, Jian Wu, Jing Xue, Lijie Wu, Rongguang Zhang & Ming Lei

  2. Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan, USA

    Jing Huang & Ming Lei

  3. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan, USA

    Jing Huang & Ming Lei

  4. Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona, USA

    Andrew F Brown, Christopher J Bley, Dustin P Rand & Julian J-L Chen

Authors
  1. Jing Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew F Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jian Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jing Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christopher J Bley
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dustin P Rand
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lijie Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rongguang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Julian J-L Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ming Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.H. carried out the bulk of the experiments; C.J.B. and D.P.R. established constructs and protocols for expression and purification of the soluble telomerase monomeric protein–RNA core complex; A.F.B. performed the telomerase assays; J.W., L.W. and R.Z. determined the CR4/5–TRBD structure; J.X. purified the S. pombe TRBD protein; J.J.-L.C. and M.L. designed the project and wrote the paper.

Corresponding authors

Correspondence to Julian J-L Chen or Ming Lei.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Structure of the TRBD–CR4/5 complex.

(a) ITC measurement of the interaction between medaka TRBD (residues 318-579) and CR4/5 (nt 170-220). (b) Electron density map of the TRBD-CR4/5 interface. 2Fo-Fc map of the CR4/5 nucleotides (orange) and the TRBD residues (blue) at the TRBD-CR4/5 interface are contoured at 1.5 σ, related to Fig. 3c,d,f and Supplementary Fig. 3b,c. (c) Superposition of the two sets of TRBD-CR4/5 molecules in the asymmetric unit. The helices α4 and α9 in the molecules A have an altered orientation due to crystal packing. (d) Structural superposition of the Oryzias latipes (medaka) TRBD-CR4/5 complex (blue) with the Takifugu rubripes (fugu) TRBD domain (orange, PDB accession code 4LMO1).

Supplementary Figure 2 Sequence alignments of the TRBD domain of TERT and the CR4/5 (TWJ) domain of TR.

(a) Primary sequence alignment of the TRBD domain of TERT. Conserved motifs of TRBD are boxed. Secondary structure assignments are shown as cylinders (α helices) and arrows (β strands) and are colored blue. Ol_Tert, Oryzias latipes; Dr_Tert, Danio rerio; Hs_Tert, Homo sapiens; Mm_Tert, Mus musculus; Xl_Tert, Xenopus laevis; Tc_Tert, Tribolium castaneum; Tt_Tert, Tetrahymena thermophila; Sp_Tert, Schizosaccharomyces pombe. (b) Sequence alignment of the vertebrate CR4/5 domains and the fungi TWJ domains, based on predicted secondary structures. Conserved RNA secondary structures are denoted. The conserved nucleotide A at the junction J6/J6.1 as well as its interacting nucleotide G are highlighted in orange.

Supplementary Figure 3 Structural and mutational analyses of the TRBD-CR4/5 interaction.

(a) Structural superposition of the crystal structure of medaka P6.1 (orange) with the NMR structures of human P6.1 (cyan, PDB accession code 1OQ02) and pseudouridylated human P6.1 (magenta, PDB accession code 2KYE3). (b) Stereo view of the stem P6-mediated TRBD-CR4/5 interactions. The intermolecular hydrogen bonds are shown as dashed magenta lines. (c) Stereo view of the stem P6.1-mediated TRBD-CR4/5 interactions. (d) ITC measurements of the interactions between wild-type and mutant TRBD and CR4/5, related to Table 2. ol_TRBD, Oryzias latipes TRBD; sp_TWJ, Schizosaccharomyces pombe three-way junction.

Supplementary Figure 4 Analyses of the TRBD-CR4/5 and TRBD-TWJ interactions.

(a) Superposition of the TRBD-CR4/5 complex structure with the NMR structure of the medaka CR4/5 based on stem P6 (left panel) and stem P6.1 (right panel), respectively. TRBD is colored blue and the CR4/5 free and in the complex is colored cyan and orange, respectively. In the structure of the free CR4/5, the TRBD-binding nucleotides on stem P6 and stem P6.1 are colored magenta and yellow, respectively. The black box denotes the spatial collision happens between TRBD and stem P5 in the structural superposition based on stem P6.1. (b) ITC measurements of the interactions between wild-type and the A1060 mutants of S. pombe TWJ and TRBD, related to Table 3. The TWJ_A1060G mutant RNA was not correctly folded, as shown in its gel filtration profile compared with that of the wild-type TWJ RNA. (c) Telomerase primer-extension assays of wild type and mutant S. pombe and N. crassa telomerases, related to Fig. 4c. (d) Fugu CR4/5 can bind to medaka TRBD efficiently, as shown in the HiLoad 200 gel filtration profile.

Supplementary Figure 5 Implication of the TRBD–CR4/5 structure on the architecture of vertebrate telomerase.

(a) Comparison of the structural organizations between the distal SL4 region of ciliate telomerase RNA (left panel) and the stem P6.1 of vertebrate CR4/5 (right panel). The organization of SL4 in Tetrahymena telomerase is adapted from Fig. 2g in Ref. 27 of the main text. In both structures, TRBD is shown as electrostatic potential surface. The medaka CR4/5-TRBD structure is oriented in the same direction as that of the Tetrahymena TRBD. (b) A proposed architecture of vertebrate telomerase based on the CR4/5-TRBD crystal structure, shown in two perpendicular views. The color theme and the figure denotation are the same as that of Fig. 1a in the main text.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 988 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huang, J., Brown, A., Wu, J. et al. Structural basis for protein-RNA recognition in telomerase. Nat Struct Mol Biol 21, 507–512 (2014). https://doi.org/10.1038/nsmb.2819

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2819

This article is cited by

Search

Advanced 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