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Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA

Nature Structural & Molecular Biology volume 15, pages 34–42 (2008)Cite this article

Abstract

Although the catalytic subunit of the Schizosaccharomyces pombe telomerase holoenzyme was identified over ten years ago, the unusual heterogeneity of its telomeric DNA made it difficult to identify its RNA component. We used a new two-step immunoprecipitation and reverse transcription–PCR technique to identify the S. pombe telomerase RNA, which we call TER1. TER1 RNA was 1,213 nucleotides long, similar in size to the Saccharomyces cerevisiae telomerase RNA, TLC1. TER1 RNA associated in vivo with the two known subunits of the S. pombe telomerase holoenzyme, Est1p and Trt1p, and neither association was dependent on the other holoenzyme component. We present a model to explain how telomerase introduces heterogeneity into S. pombe telomeres. The technique used here to identify TER1 should be generally applicable to other model organisms.

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Figure 1: Loss of viability and telomeric DNA in cells lacking the candidate gene for telomerase RNA.
Figure 2: Expression of a ter1 allele with a mutated template promotes the incorporation of altered repeats into native telomeres.
Figure 3: TER1 RNA is ∼1,200 nt.
Figure 4: Predicted secondary structure for TER1 RNA.
Figure 5: Disruption of the TER1 template-proximal helix causes telomeric shortening and increased incorporation of the infrequently added terminal cytosine, which can be rescued by a compensatory helix restoration mutant.
Figure 6: TER1 RNA is associated with the holoenzyme components Trt1p and Est1p in vivo, but not with Pku80p.
Figure 7: Model for interaction between the S. pombe telomeric DNA and TER1 RNA and the addition of telomeric sequence.

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Acknowledgements

We thank P. Baumann for sharing results before publication and J.B. Boulé, M. Mateyak, J. Phillips, S. Pinter, M. Sabourin, C. Tuzon and Y. Wu for critical reading of the manuscript. We thank O. Troyanskya and C. Huttenhower for computational assistance, R. Allshire (Wellcome Trust Centre for Cell Biology), P. Baumann (Stowers Institute for Medical Research), H. Lieberman (Columbia University) and M. Sipiczki (University of Debrecen) for strains, and K. Miller in J. Cooper's lab, C. Tuzon and J. Bruzik for protocols. This work was supported by US National Institutes of Health grants GM43265 and R37 GM26938 and by a US National Research Service Award to C.J.W.

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  1. Department of Molecular Biology, Princeton University, Princeton, 08544, New Jersey, USA

    Christopher J Webb & Virginia A Zakian

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Correspondence to Virginia A Zakian.

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Webb, C., Zakian, V. Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA. Nat Struct Mol Biol 15, 34–42 (2008). https://doi.org/10.1038/nsmb1354

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