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The finger subdomain of yeast telomerase cooperates with Pif1p to limit telomere elongation

Nature Structural & Molecular Biology volume 13, pages 734–739 (2006)Cite this article

Abstract

Telomere synthesis depends on telomerase, which contains an RNA subunit linked to a specialized reverse transcriptase subunit and several associated proteins. Here we report the characterization of four mutations in the yeast reverse transcriptase subunit Est2p that cause an overelongation of telomeres and an increase in the association of Est1p with telomeres during S phase. These 'up-mutations' are clustered in the finger subdomain of the reverse transcriptase. We show that the catalytic properties of the up-mutant telomerases are not improved in vitro. In vivo, the up-mutations neither bypass the activation step governed by Cdc13p nor do they uncouple telomerase from the Rap1p inhibition pathway. In the presence of the up-mutations, however, the ability of the Pif1p helicase to decrease telomere length and to inhibit the association of Est1p with telomeres is impaired. In addition, Pif1p associates in vivo with the telomerase RNA (TLC1) in a way that depends on the finger subdomain. We propose that, in addition to its catalytic role, the finger subdomain of Est2p facilitates the action of Pif1p at telomeres.

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Figure 1: Overelongation of telomeric DNA in est2-up cells.
Figure 2: Telomeric association of Est1p-myc in wild-type, pif1Δ, est2-up34 and est2-up34 pif1Δ strains.
Figure 3: est2-up mutations cooperate with PIF1 to regulate telomere length.
Figure 4: The genetic interaction between est2 up-mutations and PIF1 is highly specific.
Figure 5: TLC1 mRNA is enriched in Pif1p-immunoprecipitated fractions in presence of est2-up34, but not wild-type EST2.

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Acknowledgements

We thank V. Zakian, J. Lopez, C. Ribeyre, A. Nicolas, D. Gottschling, T. Petes, D. Shore, M. Hall, M. Bickle, F. Letourneur and V. Lundblad for plasmids, strains and antibodies, A. Nicolas for critically reading the manuscript and T. Teixeira and B. Canard for discussion and advice. Work in E.G.'s and V.G.'s laboratories was supported by La Ligue Nationale contre le Cancer (Equipes labellisées); E.G.'s laboratory was also funded by European Contract FI6R-CT-2003-58842, named RISC-RAD; work in J.F.P.'s laboratory was supported by the CNR Programma biomolecole per la salute umana and COFIN-2001 (MURST); work in J.L.'s laboratory was supported by the Swiss National Science Foundation; C.L. was supported by a fellowship cofinanced by the Fondo Sociale Europeo and by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica; and work by C.L. was done in fulfillment of the requirements for a joint doctoral degree in Genetics at the University of Naples 'Federico II' and at the Ecole Normale Supérieure (Lyon).

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Author notes

  1. Anne Eugster and Chiara Lanzuolo: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratoire de Biologie Moléculaire et Cellulaire, UMR5161, IFR128, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, Lyon, 69364 Cedex 07, France

    Anne Eugster, Chiara Lanzuolo, Manon Bonneton, Emma Stegberg, Anne-Sophie Berthiau & Eric Gilson

  2. Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli Federico II, via Cinthia 26, Napoli, 80126, Italy

    Chiara Lanzuolo, Alessandra Pollice & John F Pulitzer

  3. Laboratoire d'Instabilité Génétique et Cancérogenèse, CNRS, 31 Chemin Joseph Aiguier, Marseille, 13402 Cedex 20, France

    Pierre Luciano, Yves Corda & Vincent Géli

  4. Swiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne and NCCR Program 'Frontiers in Genetics,', Chemin des Boveresses 155, Epalinges, CH-1066, Switzerland

    Klaus Förstemann & Joachim Lingner

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  1. Anne Eugster
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Contributions

A.E., C.L., M.B., P.L., E.S., A.-S.B., K.F. and Y.C. did and interpreted the experiments. A.P., J.L. and J.F.P. contributed to the design and interpretation of the experiments. V.G. and E.G. coordinated the whole work and cowrote the paper.

Corresponding author

Correspondence to Eric Gilson.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Expression of Est2p is not altered by the est2-up mutations (PDF 96 kb)

Supplementary Fig. 2

The est2-up mutations act downstream of telomerase recruitment (PDF 869 kb)

Supplementary Methods (PDF 157 kb)

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Eugster, A., Lanzuolo, C., Bonneton, M. et al. The finger subdomain of yeast telomerase cooperates with Pif1p to limit telomere elongation. Nat Struct Mol Biol 13, 734–739 (2006). https://doi.org/10.1038/nsmb1126

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