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Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence

Nature Structural & Molecular Biology volume 20, pages 1199–1205 (2013)Cite this article

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Abstract

Although telomeres are heterochromatic, they are transcribed into noncoding telomeric repeat–containing RNA (TERRA). Here we show that RNA-DNA hybrids form at telomeres and are removed by RNase H enzymes in the budding yeast, Saccharomyces cerevisiae. In recombination-competent telomerase mutants, telomeric RNA-DNA hybrids promote recombination-mediated elongation events that delay the onset of cellular senescence. Reduction of TERRA and telomeric RNA-DNA–hybrid levels diminishes rates of recombination-mediated telomere elongation in cis. Overexpression of RNase H decreases telomere recombination rates and accelerates senescence in recombination-competent but not recombination-deficient cells. In contrast, in the absence of both telomerase and homologous recombination, accumulation of telomeric RNA-DNA hybrids leads to telomere loss and accelerated rates of cellular senescence. Therefore, the regulation of TERRA transcription and telomeric RNA-DNA–hybrid formation are important determinants of both telomere-length dynamics and proliferative potential after the inactivation of telomerase.

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Figure 1: Telomeric RNA-DNA hybrids delay senescence in RNase H mutants.
Figure 2: Telomeric RNA-DNA hybrids promote Rad52-dependent recombination.
Figure 3: The modified telomere 7L* has reduced TERRA levels.
Figure 4: Reduced TERRA levels at 7L* hinder recombination in cis.
Figure 5: Overexpressed RNH1 hinders telomeric recombination in presenescent cells.
Figure 6: TERRA forms telomeric RNA-DNA hybrids that can lead to telomere extension or loss.

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Acknowledgements

We acknowledge M. Chang for help with alignments and T. Schmidt and M.G. Montana for strain generation. B.B. was supported by a Landesgraduiertenförderung fellowship. S.L.-G. is supported by a long-term European Molecular Biology Organization–Marie Curie cofund fellowship (ALTF 9-2010). This work was supported by the Deutsche Forschungsgemeinschaft (SFB 1036) to B.L. and the Netzwerk Alters-Forschung (Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg) to B.L.

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  1. Bettina Balk and André Maicher: These authors contributed equally to this work.

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  1. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ)-ZMBH Alliance, Heidelberg, Germany

    Bettina Balk, André Maicher, Martina Dees, Julia Klermund, Sarah Luke-Glaser, Katharina Bender & Brian Luke

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Contributions

B.B., A.M. and B.L. designed experiments. B.B., A.M., M.D., J.K., S.L.-G. and K.B. carried out experimental work and performed the data analysis. B.B., A.M., J.K., S.L.-G. and B.L. wrote the manuscript. B.L. guided the project.

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Integrated supplementary information

Supplementary Figure 1 Telomeric RNA-DNA hybrids are regulated by RNase H enzymes

RNA-DNA hybrids exist at telomeres and accumulate in RNase H mutants. The identical ChIP as described in Fig. 1a, however quantified showing % input on the y axis. Values are represented as % input of telomeric DNA recovered and are the means of seven (+ab) and five (-ab) biological replicates, error bars depict ± s.e.m.P values were derived from two-tailed Student's t-tests (NS = not significant). (b) The RNA-DNA hybrid signal is RNase H sensitive. Following overnight immunoprecipitation with the S9.6 antibodies, one wild type sample was treated with recombinant RNase H before the washing steps (see online methods). (c) TERRA levels are not increased in rnh1 rnh201 mutants. qRT-PCR was performed for the 6Y', 1L and 15L telomeres using the indicated mutants (at approx. PD 15 following tetrad dissection). sir2 cells served as positive control where TERRA is upregulated. Average values were derived from 3 biological replicates where error bars depict ± s.e.m.

Supplementary Figure 2 Accumulation of RNA-DNA hybrids promotes homologous recombination

(a) Raw data from Fig. 1b. The individual pairs within a graph were derived from the same tetrad for direct comparisons. (b) The second and third biological replicates from Fig. 2a. (c) Premature survivor formation does not account for the increased rate of HR in rnh1 rnh201 cells. Genomic DNA derived from tetrad 2 of the curve shown in Fig. 1b was digested with Xho1. TRFs of Y' telomeres can be seen between 1164 and 992 bps. Recombination rates for Fig. 2a were determined at around PD 35, when type II survivors had clearly not yet formed.

Supplementary Figure 3 Rnh1 and Rnh201 act redundantly to remove telomeric RNA-DNA hybrids

(a) The increased rate of senescence shown in Fig. 2c only occurs in absence of telomerase. Senescence curves were performed as previously described (Fig. 1b). The loss of RNase H activity does not result in decreased cell viability in rad52 cells, but accelerates senescence only when both EST2 and RAD52 are co-deleted. Curve averages were derived from 6 biological replicates for each genotype depicted, error bars depict ± s.e.m. est2 rad52 and est2 rad52 rnh1 rnh201 curves are taken form Fig. 2c. (b, c) Senescence curves in an est2 rad52 background were performed with only one of the RNase H genes deleted (rnh1 or rnh201, respectively). Rates of senescence were not affected upon deletion of one RNase H gene only. Curves represent the means of 6 biological replicates ± s.e.m. (d) Raw data from Fig. 2c. Genotypes are indicated.

Supplementary Figure 4 The THO mutant hpr1 shows increased telomeric RNA-DNA hybrids and increased rates of telomeric recombination

(a) hpr1 shows increased telomeric RNA-DNA hybrids. ChIP was carried out as described in Fig. 1a. The values are represented as % input of telomeric DNA recovered relative to wild type (set to 1) and are the means of 5 biological replicates, error bars depict ± s.e.m. P values were derived from a two-tailed one-sample Student's t-test. (b) Deletion of HPR1 accelerates senescence in HR deficient telomerase mutants. In the presence of RAD52 the viability loss was alleviated, suggesting HR-mediated compensation. Liquid senescence assays were performed on the indicated strains. Curves represent the average value for 6 biological replicates ± s.e.m. (c) hpr1 shows increased rates of telomeric recombination. 1L Telomere-PCR was performed on genomic DNA extracted from the two indicated clones, which were derived form the same tetrad. 1L Telomere-PCR products were cloned and sequenced as described in Fig. 2a and % of diverged telomeres was calculated (est2 n = 48, PD 30; est2 hpr1 n = 39; PD 26).

Supplementary Figure 5 Overexpression of RNH1 increases senescence in recombination-competent cells

(a) Raw data for Fig. 5a for the indicated genotypes. (b) Raw data for Fig. 5e for the indicated genotypes

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Balk, B., Maicher, A., Dees, M. et al. Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence. Nat Struct Mol Biol 20, 1199–1205 (2013). https://doi.org/10.1038/nsmb.2662

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