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TERRA promotes telomere shortening through exonuclease 1-mediated resection of chromosome ends.

Pfeiffer V, Lingner J - PLoS Genet. (2012)

Bottom Line: TERRA interacts with the Exo1-inhibiting Ku70/80 complex, and deletion of EXO1 but not MRE11 fully suppressed the TERRA-mediated short telomere phenotype in presence and absence of telomerase.Thus TERRA transcription facilitates the 5'-3' nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate.Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Frontiers in Genetics National Center of Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT
The long noncoding telomeric repeat containing RNA (TERRA) is expressed at chromosome ends. TERRA upregulation upon experimental manipulation or in ICF (immunodeficiency, centromeric instability, facial anomalies) patients correlates with short telomeres. To study the mechanism of telomere length control by TERRA in Saccharomyces cerevisiae, we mapped the transcriptional start site of TERRA at telomere 1L and inserted a doxycycline regulatable promoter upstream. Induction of TERRA transcription led to telomere shortening of 1L but not of other chromosome ends. TERRA interacts with the Exo1-inhibiting Ku70/80 complex, and deletion of EXO1 but not MRE11 fully suppressed the TERRA-mediated short telomere phenotype in presence and absence of telomerase. Thus TERRA transcription facilitates the 5'-3' nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate. Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities.

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1L TERRA expression depends on the Dox concentration and does not affect other TERRA species.(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 Materials and Methods 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 [33]. 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.
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pgen-1002747-g002: 1L TERRA expression depends on the Dox concentration and does not affect other TERRA species.(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 Materials and Methods 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 [33]. 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.

Mentions: We measured 1L-derived TERRA levels by quantitative reverse transcription PCR (qRT-PCR) in a wild type strain, control strains (two independently generated strains) containing a cassette lacking the TetO7 and CYC1 sequences (Figure 1A), and TetO7-1L strains (two independently generated strains) containing the TetO7 CYC1-cassette in absence and presence of different concentrations of Dox (Figure 2A). In absence of Dox, TERRA 1L was induced >200 fold in the two TetO7-1L transgenic strains. At 1 µg/ml Dox, the induction was around 30 fold and at 10 µg/ml, TERRA 1L was similar in wild type, control and TetO7-1L strains. Under induced conditions (0, 0.1 µg/ml Dox), TERRA 1L was also detectable on Northern blots (Figure 2B, lanes 3, 4, 7, 8). The transcript size ranged between 500 and 800 nt, which we deduce corresponds to a subtelomeric part of 408 nt (Figure 1A, 1C) and a telomeric UG-tract of at least 100 nt. Levels and length of induced 1L TERRA expression are comparable to total TERRA seen in a sir3Δ strain (Figure 2C, compare lanes 3 and 4 with 12 and 14; Figure S1). Deletion of SIR3 induces TERRA mostly from X-only telomeres but not from telomeres containing also Y′ elements [33]. In contrast the rat1-1 mutation leads to TERRA upregulation from all chromosome ends including Y′ element containing telomeres, giving rise to shorter telomeric transcripts that are also detected on the Northern blot (Figure 2C, lane 1). This is consistent with the position of the transcriptional start site of TERRA transcribed from Y′ element containing telomeres (8L, 8R, 12L-YP1, 12R-YP2, 13L, 15R), which is close to the 3′end of the Y′ ORF as determined by 5′RACE (Figure S2). TERRA transcribed from telomere 8R contains 173 nt of subtelomeric sequence (Figure S2B), which is considerably less than the subtelomeric sequence transcribed at the X-only TERRA 1L (346 nt). We also determined by qRT-PCR whether upregulation of TERRA from TetO7-1L would impact on TERRA levels from other chromosome ends (Figure 2D). 7L and 15L TERRA correspond like 1L to X-only containing telomeres, while 6*Y′ TERRA designates a population of TERRA molecules transcribed from 6 different Y′ element containing telomeres. Full induction of 1L TERRA in absence of Dox did not alter TERRA levels from any other chromosome end tested.


TERRA promotes telomere shortening through exonuclease 1-mediated resection of chromosome ends.

Pfeiffer V, Lingner J - PLoS Genet. (2012)

1L TERRA expression depends on the Dox concentration and does not affect other TERRA species.(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 Materials and Methods 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 [33]. 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.
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pgen-1002747-g002: 1L TERRA expression depends on the Dox concentration and does not affect other TERRA species.(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 Materials and Methods 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 [33]. 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.
Mentions: We measured 1L-derived TERRA levels by quantitative reverse transcription PCR (qRT-PCR) in a wild type strain, control strains (two independently generated strains) containing a cassette lacking the TetO7 and CYC1 sequences (Figure 1A), and TetO7-1L strains (two independently generated strains) containing the TetO7 CYC1-cassette in absence and presence of different concentrations of Dox (Figure 2A). In absence of Dox, TERRA 1L was induced >200 fold in the two TetO7-1L transgenic strains. At 1 µg/ml Dox, the induction was around 30 fold and at 10 µg/ml, TERRA 1L was similar in wild type, control and TetO7-1L strains. Under induced conditions (0, 0.1 µg/ml Dox), TERRA 1L was also detectable on Northern blots (Figure 2B, lanes 3, 4, 7, 8). The transcript size ranged between 500 and 800 nt, which we deduce corresponds to a subtelomeric part of 408 nt (Figure 1A, 1C) and a telomeric UG-tract of at least 100 nt. Levels and length of induced 1L TERRA expression are comparable to total TERRA seen in a sir3Δ strain (Figure 2C, compare lanes 3 and 4 with 12 and 14; Figure S1). Deletion of SIR3 induces TERRA mostly from X-only telomeres but not from telomeres containing also Y′ elements [33]. In contrast the rat1-1 mutation leads to TERRA upregulation from all chromosome ends including Y′ element containing telomeres, giving rise to shorter telomeric transcripts that are also detected on the Northern blot (Figure 2C, lane 1). This is consistent with the position of the transcriptional start site of TERRA transcribed from Y′ element containing telomeres (8L, 8R, 12L-YP1, 12R-YP2, 13L, 15R), which is close to the 3′end of the Y′ ORF as determined by 5′RACE (Figure S2). TERRA transcribed from telomere 8R contains 173 nt of subtelomeric sequence (Figure S2B), which is considerably less than the subtelomeric sequence transcribed at the X-only TERRA 1L (346 nt). We also determined by qRT-PCR whether upregulation of TERRA from TetO7-1L would impact on TERRA levels from other chromosome ends (Figure 2D). 7L and 15L TERRA correspond like 1L to X-only containing telomeres, while 6*Y′ TERRA designates a population of TERRA molecules transcribed from 6 different Y′ element containing telomeres. Full induction of 1L TERRA in absence of Dox did not alter TERRA levels from any other chromosome end tested.

Bottom Line: TERRA interacts with the Exo1-inhibiting Ku70/80 complex, and deletion of EXO1 but not MRE11 fully suppressed the TERRA-mediated short telomere phenotype in presence and absence of telomerase.Thus TERRA transcription facilitates the 5'-3' nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate.Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Frontiers in Genetics National Center of Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT
The long noncoding telomeric repeat containing RNA (TERRA) is expressed at chromosome ends. TERRA upregulation upon experimental manipulation or in ICF (immunodeficiency, centromeric instability, facial anomalies) patients correlates with short telomeres. To study the mechanism of telomere length control by TERRA in Saccharomyces cerevisiae, we mapped the transcriptional start site of TERRA at telomere 1L and inserted a doxycycline regulatable promoter upstream. Induction of TERRA transcription led to telomere shortening of 1L but not of other chromosome ends. TERRA interacts with the Exo1-inhibiting Ku70/80 complex, and deletion of EXO1 but not MRE11 fully suppressed the TERRA-mediated short telomere phenotype in presence and absence of telomerase. Thus TERRA transcription facilitates the 5'-3' nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate. Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities.

Show MeSH
Related in: MedlinePlus