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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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Ku70/80 binds TERRA and ku70Δ suppresses the shortening of telomere 1L upon 1L TERRA expression.(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 Materials and Methods). 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).
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pgen-1002747-g005: Ku70/80 binds TERRA and ku70Δ suppresses the shortening of telomere 1L upon 1L TERRA expression.(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 Materials and Methods). 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).

Mentions: The Ku70/80 dimer binds to telomeres [16] and has also been characterized as RNA binding protein interacting with the telomerase RNA moiety TLC1 in yeast [39], [40]. Therefore, we wondered if Ku70/80 may also bind TERRA. We tagged endogenous Ku80 at its C-terminus with three HA-tags including eight glycine residues as a linker and immunoprecipated Ku80-HA via the tag from crosslinked extracts with similar efficiency in presence or absence of TERRA induction in strain TetO7-1L (Figure 5A, upper panel, compare lanes 3 and 6). An untagged TetO7-1L strain (wt) as well as a mock IP served as negative controls. RNA was purified from crosslinked extracts and co-immunoprecipitates and presence of TERRA, Tlc1 and Act1 RNAs were measured by qRT-PCR (Figure 5A, lower panels). Act1 RNA served as negative control and was not enriched in the HA-tag fraction (Figure S7A). However, 1L TERRA was enriched more than 2.5 fold in the HA-tag fraction over a fraction obtained from the non-tagged, but fully induced TetO7-1L strain (Figure 5A, lower panel, Figure S7B). Pull-down of Tlc1 RNA served as positive control and Tlc1 RNA was enriched more than 10 fold independently of the expression of 1L TERRA (Figure 5A, lower panel). The pull-down efficiency of Tlc1 is similar to a recent publication [40]. Expression levels of Tlc1 RNA were not influenced by induction of 1L TERRA transcription (Figure S7B). We conclude that Ku70/80 binds TERRA even though the affinity for TERRA may be lower than for Tlc1 RNA. Furthermore, since we included a crosslinking step in the experiment, we cannot distinguish direct from indirect physical interactions between Ku70/80 and TERRA. However, our results indicate that TERRA and telomere transcription do not affect the interaction between Ku70/80 and Tlc1 RNA. This is in line with our previous conclusion that TERRA does not interfere with the recruitment of telomerase.


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

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

Ku70/80 binds TERRA and ku70Δ suppresses the shortening of telomere 1L upon 1L TERRA expression.(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 Materials and Methods). 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375253&req=5

pgen-1002747-g005: Ku70/80 binds TERRA and ku70Δ suppresses the shortening of telomere 1L upon 1L TERRA expression.(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 Materials and Methods). 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).
Mentions: The Ku70/80 dimer binds to telomeres [16] and has also been characterized as RNA binding protein interacting with the telomerase RNA moiety TLC1 in yeast [39], [40]. Therefore, we wondered if Ku70/80 may also bind TERRA. We tagged endogenous Ku80 at its C-terminus with three HA-tags including eight glycine residues as a linker and immunoprecipated Ku80-HA via the tag from crosslinked extracts with similar efficiency in presence or absence of TERRA induction in strain TetO7-1L (Figure 5A, upper panel, compare lanes 3 and 6). An untagged TetO7-1L strain (wt) as well as a mock IP served as negative controls. RNA was purified from crosslinked extracts and co-immunoprecipitates and presence of TERRA, Tlc1 and Act1 RNAs were measured by qRT-PCR (Figure 5A, lower panels). Act1 RNA served as negative control and was not enriched in the HA-tag fraction (Figure S7A). However, 1L TERRA was enriched more than 2.5 fold in the HA-tag fraction over a fraction obtained from the non-tagged, but fully induced TetO7-1L strain (Figure 5A, lower panel, Figure S7B). Pull-down of Tlc1 RNA served as positive control and Tlc1 RNA was enriched more than 10 fold independently of the expression of 1L TERRA (Figure 5A, lower panel). The pull-down efficiency of Tlc1 is similar to a recent publication [40]. Expression levels of Tlc1 RNA were not influenced by induction of 1L TERRA transcription (Figure S7B). We conclude that Ku70/80 binds TERRA even though the affinity for TERRA may be lower than for Tlc1 RNA. Furthermore, since we included a crosslinking step in the experiment, we cannot distinguish direct from indirect physical interactions between Ku70/80 and TERRA. However, our results indicate that TERRA and telomere transcription do not affect the interaction between Ku70/80 and Tlc1 RNA. This is in line with our previous conclusion that TERRA does not interfere with the recruitment of telomerase.

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