Telomeric transcripts stimulate telomere recombination to suppress senescence in cells lacking telomerase Tai-Yuan Yua, Yu-wen Kaob, and Jing-Jer Lina,b,1 aInstitute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan; and bInstitute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 10051, Taiwan Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved January 21, 2014 (received for review April 19, 2013) In human somatic cells or yeast cells lacking telomerase, telomeres Telomeres are transcribed from subtelomeric regions into are shortened upon each cell division. This gradual shortening of large noncoding telomeric repeat-containing RNA (TERRA) by telomeres eventually leads to senescence. However, a small RNA polymerase II. TERRA has been implicated in several population of telomerase-deficient cells can survive by bypassing telomere-related and non-telomere-related functions (11–15). senescence through the activation of alternative recombination For example, TERRA binds to telomeres and participates in pathways to maintain their telomeres. Although genes involved in regulating telomerase and the organization and maintenance of telomere recombination have been identified, mechanisms that telomeric chromatin throughout development and cellular dif- trigger telomere recombination are less known. The THO (sup- ferentiation (11–13, 16). A study using an inducible telomere pressor of the transcriptional defects of Hpr1 mutants by over- transcription system to investigate the effect of TERRA expression) complex is involved in transcription elongation and expression on telomere function (17) showed that telomeric mRNA export. Here we demonstrate that mutations in THO complex transcription causes telomere shortening in a DNA replication- components can stimulate early senescence and type II telo- dependent manner; moreover, in the absence of both telomerase mere recombination in cells lacking telomerase. The accumula- and telomere recombination, overexpression of TERRA at tion of telomere-associated noncoding telomere repeat-containing a single telomere is sufficient to induce early-onset senescence. RNA (TERRA) is required for the observed telomere effects in THO Although the cellular localization of TERRA has not been dem- complex mutants; reduced transcriptional efficiency, or overex- onstrated in yeast, RNA fluorescence in situ hybridization studies have showed that at least a fraction of TERRA molecules in pression of RNase H or C – A RNA can severely impair the type II 1 3 human and mouse cells are stably associated with telomeric telomere recombination. The results highlight a unique function for heterochromatin (11, 13). This telomeric association of TERRA telomere-associated TERRA, in the formation of type II survivors. More- may be achieved through interacting with telomeric proteins or, over, because TERRA is a long noncoding RNA, these results reveal alternatively, through the direct formation of telomeric DNA– a function for long noncoding RNA in regulating recombination. RNA hybrids. THO (a suppressor of the transcriptional defects of Hpr1 n Saccharomyces cerevisiae, the telomere length is typically mutants by overexpression) is a conserved eukaryotic complex Imaintained by the constitutive expression of telomerase. How- involved in transcription elongation and mRNA export (18, 19). ever, in cells lacking telomerase, a gradual loss of telomere In S. cerevisiae, the THO complex comprises four proteins: Hpr1, length during rounds of cell division eventually leads to the Tho2, Mft1, and Thp2 (18). In mutants with a defective THO uncapping of telomeres and induces a checkpoint-mediated cell- complex, stalled transcription and transcriptional DNA–RNA cycle arrest termed senescence (1, 2). Although most cells die in hybrids stimulate transcription-associated recombination, caus- – the absence of telomerase, rare survivors do escape senescence ing genome instability and chromosome fragility (20 23). Here, by maintaining their telomeres through a radiation sensitive 52 (RAD52)-dependent recombination mechanism (3–5). At least Significance two different types of telomeric recombination events contribute to telomere elongation in the absence of telomerase, generating Telomerase expression is essential for the long-term pro- two types of survivor cells that can be distinguished by different liferation of most cancer cells. In cancer cells lacking telomer- growth rates and distinct telomere patterns on Southern blots (3, ase, an alternative lengthening of telomeres (ALT) pathway is 6–8). Type I survivors arise through gene-conversion events that activated to maintain telomere length through telomere re- occur in the Y′ sequence elements present in the subtelomeric combination. Using yeast as a model system, we found that the BIOCHEMISTRY region of most chromosomes; these cells have short terminal noncoding telomeric repeat-containing RNA (TERRA) plays a ma- telomere repeats and grow slowly. In contrast, type II survivors jor role in recombination-mediated maintenance of telomere in have long, heterogeneous tracts of telomeric repeats, similar to telomerase-deficient cells. Increased levels of telomere-associated – the survivors identified in telomerase-deficient Kluyveromyces TERRA result in the accumulation of telomeric DNA RNA hybrids lactis and mammalian alternative lengthening of telomeres and induce telomere recombination. Because the structure and (ALT) cells (5, 9). The generation of both types of survivors and function of TERRA are conserved among eukaryotes, our find- ings suggest that the accumulation of telomere-associated the stability of their telomeres depend on the function of RAD52 TERRA might also have a role in modulating the occurrence of (3–5). However, two genetically distinct recombination pathways, ALT in cancer cells. defined by RAD50 and RAD51, govern the RAD52-dependent generation of specific types of survivors (6, 10). The type I sur- Author contributions: T.-Y.Y. and J.-J.L. designed research; T.-Y.Y. and Y.-w.K. performed vivors are absent in the rad51Δ strain, whereas type II survivors research; T.-Y.Y., Y.-w.K., and J.-J.L. analyzed data; and T.-Y.Y. and J.-J.L. wrote the paper. cannot arise in the rad50Δ strain (10). Neither type of survivor The authors declare no conflict of interest. can be found in the rad51Δ rad50Δ double mutant (6). Despite This article is a PNAS Direct Submission. the identification of these important genes that are involved in 1To whom correspondence should be addressed. E-mail: [email protected]. telomere recombination, mechanisms triggering telomere re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. combination have not been fully elucidated. 1073/pnas.1307415111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1307415111 PNAS | March 4, 2014 | vol. 111 | no. 9 | 3377–3382 Downloaded by guest on September 26, 2021 we used mutants of THO complex components to test the role of A C 10 TERRA in telomere recombination. We found that premature senescence 8 tlc1 hpr1 wt senescence and formation of RAD50-dependent type II survivors tlc1 hpr1 6 were induced in THO mutants that were also lacking functional 0 1 0 1 0 1 0 1 streaks 600 (kbp) OD 4 tlc1 telomerase. The observed premature senescence and telomere 8.0 2 tlc1 tho2 tlc1 mft1 recombination was transcription-dependent and required the 5.0 tlc1 hpr1 tlc1 thp2 accumulation of TERRA in telomeric DNA–RNA hybrids. Our 4.0 0 results have revealed a mechanism in which the accumulation 3.0 30 40 50 60 70 80 90 100 110 120 Generations of telomere-associated TERRA in THO mutants induces re- 2.0 1.5 Y’-tel tlc1 tlc1 tho2 tlc1 hpr1 combination in the absence of telomerase. More significantly, 1.0 D (kbp) we found that telomere-associated TERRA plays a role in type II 8.0 6.0 telomere recombination to facilitate the bypass of senescence in B 4.0 cells lacking telomerase. 2.0 1 1.5 2 4 1.0 Results tlc1 0.5 3 0.25 Mutations in the THO Complex Components Induce Early Senescence Restreaks Dilutions12 3 4 5 6 7 1234567 1234567 and Type II Survivor Formation in the Absence of Telomerase. Our tlc1 mft1 tlc1 thp2 previous work demonstrated that telomeres are lengthened by (kbp) tlc1 hpr1 ∼ – tho2Δ hpr1Δ tlc1 tho2 8.0 50 100 bp in or strains but remain unaffected in 6.0 mft1Δ or thp2Δ cells; moreover, mutations in these THO com- 4.0 plex components affect telomere length by reducing the expres- 2.0 1.5 sion of a telomere-associated protein, Rif1 (24). To determine 1.0 tlc1 mft1 hpr1 tho2 tlc1 thp2 0.5 whether telomere lengthening in and cells requires 0.25 telomerase activity, the telomerase RNA component-encoding Dilutions 1234567 1234567 gene TLC1 was deleted in hpr1 and tho2 cells. Lacking functional E pRS42- pRS426 RIF1 tlc1 tlc1 tho2 tlc1 hpr1 telomerase, tlc1 cells exhibited progressive shortening of telo- F (kbp) meres (Fig. 1A), whereas the lengths of Y′-bearing telomeres in 8.0 tlc1 6.0 hpr1 cells appeared ∼75 bp longer than those in wild-type cells, 4.0 as reported (24). The telomere lengths in tlc1 hpr1 cells were 2.0 1.5 similar to those in tlc1 cells, indicating that the increase of 1.0 pRS426 telomere lengths observed in hpr1 and tho2 cells required func- 0.5 0.25 tional telomerase. Interestingly, the type II survivor pattern was tlc1 tho2 frequently observed in tlc1 hpr1 and tlc1 tho2 double-mutant cells 8.0 after restreaking, but not in wild-type or single-mutant cells (Fig. 6.0 RIF1 1A and Fig. S1). The results suggest that the THO complex might 4.0 - tlc1 hpr1 2.0 somehow affect telomere recombination. 1.5 1.0 1 pRS426 To determine the role of the THO complex in senescence and 0.5 cell survival in the absence of telomerase, we examined the 2 4 0.25 growth of tlc1 cells with THO mutations. Cells were harvested 3 Restreaks Dilutions 12345678 12345678 12345678 directly from freshly dissected tlc1 spores and assayed by suc- HPR1 THO2 cessive restreaking of grown colonies onto fresh plates four Fig.