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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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Development of an inducible 1L TERRA expression system.(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 [44]. 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.
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pgen-1002747-g001: Development of an inducible 1L TERRA expression system.(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 [44]. 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.

Mentions: We mapped the transcriptional start site of TERRA at chromosome 1L (Figure 1A, 1B) by rapid amplification of 5′ ends (5′RACE) of capped RNAs in S. cerevisiae strain S288C sir3Δ. The 5′RACE was facilitated by deletion of SIR3 as Sir3 represses telomere transcription [33]. 5′ RACE products were homogeneous in size (Figure 1B, lane 2) and cloning and sequencing of 5′ RACE products revealed existence of one major start site 346 nucleotides (nt) upstream of the TG-telomeric repeats, close to the telomere-proximal end of the X-core element present at 1L (Figure 1A and 1C).


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

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

Development of an inducible 1L TERRA expression system.(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 [44]. 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.
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pgen-1002747-g001: Development of an inducible 1L TERRA expression system.(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 [44]. 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.
Mentions: We mapped the transcriptional start site of TERRA at chromosome 1L (Figure 1A, 1B) by rapid amplification of 5′ ends (5′RACE) of capped RNAs in S. cerevisiae strain S288C sir3Δ. The 5′RACE was facilitated by deletion of SIR3 as Sir3 represses telomere transcription [33]. 5′ RACE products were homogeneous in size (Figure 1B, lane 2) and cloning and sequencing of 5′ RACE products revealed existence of one major start site 346 nucleotides (nt) upstream of the TG-telomeric repeats, close to the telomere-proximal end of the X-core element present at 1L (Figure 1A and 1C).

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