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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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Exo1 mediates shortening of telomere 1L upon 1L TERRA expression, while Mre11 is not involved.(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.
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pgen-1002747-g006: Exo1 mediates shortening of telomere 1L upon 1L TERRA expression, while Mre11 is not involved.(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.

Mentions: Telomere shortening is the consequence of incomplete telomere end replication and the action of several nucleases which process chromosome ends in order to generate 3′ overhangs (see Introduction). Notably, Ku70/80 function includes protection of chromosome ends from excessive degradation by the 5′-3′ exonuclease Exo1. In order to test if TERRA transcription promotes nuclease attack at chromosome ends, we determined if deletion of known chromosome end processing activities abolished TERRA-induced telomere shortening. Significantly, deletion of EXO1 rescued the short telomere phenotype observed at TetO7-1L after TERRA induction (Figure 6A, compare −Dox with +Dox lanes). On the other hand, exo1Δ did not notably influence telomere length of 1L in the absence of induced transcription (Figure 6A, compare lanes 2, 4, 6, 8 to lanes 10, 12, 14, 16; Figure S9B, S9C) nor did exo1Δ affect length of telomere 6R (Figure 6B). Furthermore, TERRA TetO7-1L expression was not suppressed in exo1Δ (Figure S3F), although the overall TERRA levels in the exo1Δ strain were slightly decreased in comparison to wt strains (Figure S9A). Moreover, telomere 1L transcription did not induce a global DNA damage response (DDR) in TetO7-1L and the exo1Δ/TetO7-1L strains as determined by quantification of Rnr3 mRNA levels (Figure S9A).


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

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

Exo1 mediates shortening of telomere 1L upon 1L TERRA expression, while Mre11 is not involved.(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.
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pgen-1002747-g006: Exo1 mediates shortening of telomere 1L upon 1L TERRA expression, while Mre11 is not involved.(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.
Mentions: Telomere shortening is the consequence of incomplete telomere end replication and the action of several nucleases which process chromosome ends in order to generate 3′ overhangs (see Introduction). Notably, Ku70/80 function includes protection of chromosome ends from excessive degradation by the 5′-3′ exonuclease Exo1. In order to test if TERRA transcription promotes nuclease attack at chromosome ends, we determined if deletion of known chromosome end processing activities abolished TERRA-induced telomere shortening. Significantly, deletion of EXO1 rescued the short telomere phenotype observed at TetO7-1L after TERRA induction (Figure 6A, compare −Dox with +Dox lanes). On the other hand, exo1Δ did not notably influence telomere length of 1L in the absence of induced transcription (Figure 6A, compare lanes 2, 4, 6, 8 to lanes 10, 12, 14, 16; Figure S9B, S9C) nor did exo1Δ affect length of telomere 6R (Figure 6B). Furthermore, TERRA TetO7-1L expression was not suppressed in exo1Δ (Figure S3F), although the overall TERRA levels in the exo1Δ strain were slightly decreased in comparison to wt strains (Figure S9A). Moreover, telomere 1L transcription did not induce a global DNA damage response (DDR) in TetO7-1L and the exo1Δ/TetO7-1L strains as determined by quantification of Rnr3 mRNA levels (Figure S9A).

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