(A) Scheme of telomere 1L in wild type, the TetO7-1L and the control strains. +1 in the wt marks the transcription start site of 1L TERRA mapped in sir3Δ (see B, C). Numbers give the distance of the +1 site, the X-core element, or the URA3 marker from the TG repeat sequence (TG_(1–3)). The arrows towards the right mark the position of the subtelomeric oligonucleotide (oBL1180, see C) used for telomere PCR of telomere 1L. Strain TetO7-1L was constructed by introducing a sequence containing URA3, the ADH1-terminator (Ter), seven TetO boxes (TetO7) and a cytochrome 1 (CYC1) sequence upstream of the 1L TERRA transcription start site. +1 marks the transcription start site of the inducible 1L TERRA in strain TetO7-1L (see C, D). Presence of Dox inhibits 1L TERRA expression in TetO7-1L. The control strain was constructed by insertion of the URA3 cassette and the ADH1-terminator upstream of the +1 start site, without the TetO7 and CYC1 promoter sequences. (B) Nested PCR of 5′RACE (absence (−) or presence (+) of Tobacco Acid Pyrophosphatase (TAP): removes the 5′ cap structure of the RNA) performed in sir3Δ for mapping the transcription start site (+1) of 1L TERRA. Marker (M) is given in base pairs. (C) Sequence of telomere 1L in strain TetO7-1L. The CYC1 sequence upstream of the +1 start site of 1L TERRA in wt (bold and underlined) is shown in italics. The putative TATA boxes of the CYC1 sequence are underlined . The +1 start site of 1L TERRA in TetO7-1L is shown in bold, and underlined. The X-core sequence is highlighted in grey. Marked are the oligonucleotides used for the RT of the 5′RACE (oBL1181; antisense strand), for telomere PCR of telomere 1L (oBL1180; sense strand), for the nested PCR (oNI55; antisense strand), and for 1L TERRA detection by qRT-PCR (oNI54 (sense strand), oBL1181) in the X-repeat sequence (downstream of the X-core sequence). (D) Nested PCR of 5′RACE (as in B) performed on two independently generated clones of strain TetO7-1L for mapping the transcriptional start site (+1) of 1L TERRA.

(A) 1L TERRA expression can be modulated by the amount of Dox. qRT-PCR analysis of 1L TERRA in wt, TetO7-1L (two independent clones: cl1 and cl2), and control strains (two independent clones: cl1 and cl2) grown with different Dox concentrations to exponential phase at 30°C in rich medium. −ΔΔCT values of strains grown in 1, 0.1, 0 µg/ml Dox, normalized against actin with standard deviations are shown. The −ΔΔCT values corresponding to each strain grown in 10 µg/ml Dox is arbitrarily set to 0. (for calculations see published online). (B) Expressed 1L TERRA has a size of 500–800 nt. RNA was extracted from the indicated strains (as in A) grown to exponential phase at 30°C in rich medium. 15 µg of RNA was loaded per lane on a 1.2% formaldehyde/agarose (FA) gel and analyzed by Northern blot analysis. The Northern blot was hybridized with a 5′ end-radiolabeled CA oligonucleotide detecting telomeric GU repeats in 1L TERRA. The 1L TERRA signal is marked with a line. An asterisk marks an unspecific band detected with this probe. RNA marker sizes are indicated at the left in nt. (C) The size of 1L TERRA expressed from TetO7-1L is comparable to the size of TERRA species expressed from other X-only telomeres in a sir3Δ mutant. RNA was extracted from the indicated strains grown to exponential phase at 30°C in rich medium. The temperature-sensitive rat1-1 mutant, wt and sir3Δ strains were grown at 25°C to exponential phase in rich medium before the culture was split and either shifted to 37°C or maintained at 25°C for 1 h followed by RNA extraction. 15 µg of RNA was analyzed by Northern blot as described in (B). TERRA transcribed from Y′ and X-only telomeres (all TERRA) are enriched in rat1-1 cells at non-permissive temperature. sir3Δ specifically increases TERRA levels transcribed from X-only telomeres, such as telomere 1L, independently of the temperature . The asterisk marks an unspecific band. RNA marker sizes are given to the left in nt. (D) 1L TERRA expression does not affect the levels of TERRA species transcribed from other telomeres. qRT-PCR analysis of TERRA transcribed from X-only telomeres 1L, 7L, 15L, or from 6 different Y′ telomeres (6*Y′). The indicated strains were grown in rich medium with (+) or without (−) Dox to exponential phase at 30°C. Average −ΔΔCT values of three independent biological replicates normalized against actin with standard deviation are shown. −ΔΔCT values of each strain grown in +Dox is arbitrarily set to 0.

(A) 1L TERRA expression leads to shortening of telomere 1L. Upper panel: DNA was extracted from five independent clones of the TetO7-1L strain grown at 30°C on YPD plates with (+) or without (−) Dox for 25 generations and analyzed by telomere PCR for telomere 1L on a 2.5% agarose gel. Marker (M) is given in bp. Lower panel: 1L telomere PCR products were TOPO cloned and sequenced. Shown is the average length of the TG-tract under inducing (−Dox) and non-inducing (+Dox) conditions with standard deviations. 10 sequences were analyzed for each condition. (B) Telomere 6R is not affected by expression of 1L TERRA. Telomere PCR for telomere 6R performed with DNA from (A). (C) 1L TERRA expression does not affect the average length of Y′ containing telomeres. DNA was extracted from the indicated strains grown as in (A) and digested with XhoI before Southern blot analysis. The Southern blot was hybridized with a 5′ end-radiolabeled CA oligonucleotide detecting telomeric TG repeats of yeast telomeres. The telomeric TG repeat signals are highlighted with a line (XhoI fragment). Marker (M) is given in bp.

(A) DNA was extracted from TetO7-1L, and est1Δ/TetO7-1L (four independent clones) strains grown for 25 generations (gen) at 25°C on YPD plates containing 10 µg/ml Dox (lanes 3, 6, 9, 12), before plating them on YPD plates with (+) (lanes 5, 8, 11, 14) or without (−) Dox (lanes 4, 7, 10, 13) for additional 25 generations. Telomere length at 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (B) 1L TERRA expression does not accelerate shortening of telomere 6R in the absence of telomerase. Telomere PCR for telomere 6R performed with DNA from the same strains and growth conditions as in (A). (C) Recruitment of Est2 is not affected by expression of 1L TERRA. Yeast strains were grown for 25 generations at 30°C on YPD plates with (+) or without (−) Dox. Est2-myc associated chromatin was immunoprecipitated after an additional growth to exponential phase in rich medium (−/+Dox) at 30°C (for details see ). The immunoprecipitated telomeres 1L, 7L and 15L were quantified by real-time PCR and expressed as percentage of input. A mock IP lacking the myc-antibody served as negative control. Values of two independent biological replicates with standard deviation are shown.

(A) TERRA is associated with Ku70/80. Yeast strains were grown for 25 generations at 25°C on YPD plates with (+) or without (−) Dox. Ku80-HA associated RNA was immunoprecipitated after an additional growth to exponential phase in rich medium (−/+Dox) at 25°C (for details see ). A mock IP lacking the HA-antibody as well as an untagged strain (wt) served as controls. Upper panel: 1L TERRA expression does not influence the specific pull-down efficiency of Ku80-HA in RNA-ChIP experiments as determined by Western blot. After crosslink reversal, input (IN) and immunoprecipitates (IP) were analyzed by PAGE. The Ku80-HA protein band is highlighted by an arrow head; an unspecific band is marked by an asterisk. Marker is given in kDA. Lower panels: The immunoprecipitated 1L TERRA and Tlc1 RNA were quantified by RT-PCR and expressed relative to the untagged strains. Values of two independent biological replicates with standard deviation are shown. (B) 1L telomere shortening upon 1L TERRA expression involves Ku70. DNA was extracted from four independent clones of the indicated strains grown at 30°C for 25 generations on YPD plates with (+) or without (−) Dox. Telomere length of telomere 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (C) 1L TERRA expression does not affect the length of telomere 6R in the absence of Ku70. Telomere PCR for telomere 6R performed with DNA from (B).

(A) TERRA induced shortening of telomere 1L is abolished by deletion of exonuclease 1 (exo1Δ). DNA was extracted from four independent clones of the indicated strains grown at 30°C for 25 generations on YPD plates with (+) or without (−) Dox. Telomere length at 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (B) Length of telomere 6R is not affected by 1L TERRA expression in the absence of Exo1. Telomere PCR for telomere 6R performed with DNA from (A). (C) Deletion of Exo1 rescues increased telomere shortening of telomere 1L upon TERRA induction in the absence of telomerase. est1Δ/TetO7-1L (four independent clones) and est1Δ/exo1Δ/TetO7-1L (three independent clones) strains were grown for 25 generations at 25°C on YPD plates containing 10 µg/ml Dox, before plating them on YPD plates with (+) (lanes 2, 4, 6, 8, 10, 12, 14) or without (−) (lanes 1, 3, 5, 7, 9, 11, 13) Dox for additional 25 generations. Telomere lengths at 1L were analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (D) Telomere shortening by 1L TERRA expression is not mediated by Mre11. DNA was extracted from four independent clones of the indicated strains grown at 30°C for 25 generations on YPD plates with (+) or without (−) Dox. Telomere length at 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp.