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The heterochromatic chromosome caps in great apes impact telomere metabolism.

Novo C, Arnoult N, Bordes WY, Castro-Vega L, Gibaud A, Dutrillaux B, Bacchetti S, Londoño-Vallejo A - Nucleic Acids Res. (2013)

Bottom Line: In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species.Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution.In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres.

View Article: PubMed Central - PubMed

Affiliation: Telomeres and Cancer laboratory, 'Equipe Labellisée Ligue contre le Cancer', UMR3244, Institut Curie, 26 rue d'Ulm, 75248 Paris, France.

ABSTRACT
In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species. Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution. Here, we show that telomeres on chromosome arms carrying subtelomeric heterochromatic caps in the chimpanzee, which are completely absent in humans, replicate later than telomeres on chromosome arms without caps. In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres. Strikingly, telomere-containing RNA accumulates extrachromosomally in gorilla mitotic cells, suggesting that at least some aspects of telomere-containing RNA biogenesis have diverged in gorilla, perhaps in concert with the evolution of heterochromatic caps in this species.

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XRN2 downregulation leads to TERRA and Cht7 RNA accumulation. (A) XRN2 expression was downregulated in gorilla cells using a specific siRNA. Levels of downregulation were estimated using RT–qPCR to quantify XRN2 mRNA levels 36 and 48 h after transfection. (B) After siXRN2 treatment, total RNA was analyzed by northern blot, and TERRA molecules were revealed with a telomeric C-rich (Tel C) probe (γ-P32-labeled) (left panel). Relative to the amount of RNA loaded (rightmost panel), levels of TERRA increased in cells after 48 h of treatment with siXRN2. The same membrane was stripped and re-hybridized with a telomeric G-rich probe (γ-P32-labeled) and with a probe (γ-P32-labeled) against the Cht7 minisatellite repeat (second to right panel). RNAs containing this repeat also became more abundant in siXRN2-treated cells.
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gkt169-F4: XRN2 downregulation leads to TERRA and Cht7 RNA accumulation. (A) XRN2 expression was downregulated in gorilla cells using a specific siRNA. Levels of downregulation were estimated using RT–qPCR to quantify XRN2 mRNA levels 36 and 48 h after transfection. (B) After siXRN2 treatment, total RNA was analyzed by northern blot, and TERRA molecules were revealed with a telomeric C-rich (Tel C) probe (γ-P32-labeled) (left panel). Relative to the amount of RNA loaded (rightmost panel), levels of TERRA increased in cells after 48 h of treatment with siXRN2. The same membrane was stripped and re-hybridized with a telomeric G-rich probe (γ-P32-labeled) and with a probe (γ-P32-labeled) against the Cht7 minisatellite repeat (second to right panel). RNAs containing this repeat also became more abundant in siXRN2-treated cells.

Mentions: It has been shown in the budding yeast that the RNA exonuclease Rat1P controls the levels of yeast TERRA by inducing its degradation (32). Although the role of the Rat1P-homologue XRN2 (5′–3′ exoribonuclease 2) in controlling TERRA levels remains to be demonstrated in higher primates, we tested whether XRN2 was involved in TERRA regulation in gorilla cells. Using an siRNA approach, we achieved a partial expression knockdown of the gorilla XRN2 gene (60% as determined by RT–PCR, Figure 4A) after 48 h of treatment. Strikingly, this decrease in the expression of XRN2 was associated with an increase in the detection of TERRA molecules by northern blot (Figure 4B). Remarkably, an increase in extrachromosomal TERRA molecules was also detected during mitosis, leading to a significant increase in the total fluorescence intensity associated with metaphases from cells treated with siXRN2 when compared with cells treated with an irrelevant siRNA (Figure 5A and B). This increase completely disappeared when metaphase preparations were treated with RNase (Figure 5A and B). Although most TERRA seemed to accumulate extrachromosomally, it is possible that TERRA also accumulated in association with chromosome ends, as suggested both by the fact that the proportion of sister chromatids bearing telomeric doublets was increased in siXRN2-treated cells and by the fact that this increase was lost on RNase treatment (Figure 5C and D). At the same time, as there was not detectable difference in the overall intensity of telomeres after RNase treatment (Figure 5D), telomere replication was likely not affected.Figure 4.


The heterochromatic chromosome caps in great apes impact telomere metabolism.

Novo C, Arnoult N, Bordes WY, Castro-Vega L, Gibaud A, Dutrillaux B, Bacchetti S, Londoño-Vallejo A - Nucleic Acids Res. (2013)

XRN2 downregulation leads to TERRA and Cht7 RNA accumulation. (A) XRN2 expression was downregulated in gorilla cells using a specific siRNA. Levels of downregulation were estimated using RT–qPCR to quantify XRN2 mRNA levels 36 and 48 h after transfection. (B) After siXRN2 treatment, total RNA was analyzed by northern blot, and TERRA molecules were revealed with a telomeric C-rich (Tel C) probe (γ-P32-labeled) (left panel). Relative to the amount of RNA loaded (rightmost panel), levels of TERRA increased in cells after 48 h of treatment with siXRN2. The same membrane was stripped and re-hybridized with a telomeric G-rich probe (γ-P32-labeled) and with a probe (γ-P32-labeled) against the Cht7 minisatellite repeat (second to right panel). RNAs containing this repeat also became more abundant in siXRN2-treated cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643582&req=5

gkt169-F4: XRN2 downregulation leads to TERRA and Cht7 RNA accumulation. (A) XRN2 expression was downregulated in gorilla cells using a specific siRNA. Levels of downregulation were estimated using RT–qPCR to quantify XRN2 mRNA levels 36 and 48 h after transfection. (B) After siXRN2 treatment, total RNA was analyzed by northern blot, and TERRA molecules were revealed with a telomeric C-rich (Tel C) probe (γ-P32-labeled) (left panel). Relative to the amount of RNA loaded (rightmost panel), levels of TERRA increased in cells after 48 h of treatment with siXRN2. The same membrane was stripped and re-hybridized with a telomeric G-rich probe (γ-P32-labeled) and with a probe (γ-P32-labeled) against the Cht7 minisatellite repeat (second to right panel). RNAs containing this repeat also became more abundant in siXRN2-treated cells.
Mentions: It has been shown in the budding yeast that the RNA exonuclease Rat1P controls the levels of yeast TERRA by inducing its degradation (32). Although the role of the Rat1P-homologue XRN2 (5′–3′ exoribonuclease 2) in controlling TERRA levels remains to be demonstrated in higher primates, we tested whether XRN2 was involved in TERRA regulation in gorilla cells. Using an siRNA approach, we achieved a partial expression knockdown of the gorilla XRN2 gene (60% as determined by RT–PCR, Figure 4A) after 48 h of treatment. Strikingly, this decrease in the expression of XRN2 was associated with an increase in the detection of TERRA molecules by northern blot (Figure 4B). Remarkably, an increase in extrachromosomal TERRA molecules was also detected during mitosis, leading to a significant increase in the total fluorescence intensity associated with metaphases from cells treated with siXRN2 when compared with cells treated with an irrelevant siRNA (Figure 5A and B). This increase completely disappeared when metaphase preparations were treated with RNase (Figure 5A and B). Although most TERRA seemed to accumulate extrachromosomally, it is possible that TERRA also accumulated in association with chromosome ends, as suggested both by the fact that the proportion of sister chromatids bearing telomeric doublets was increased in siXRN2-treated cells and by the fact that this increase was lost on RNase treatment (Figure 5C and D). At the same time, as there was not detectable difference in the overall intensity of telomeres after RNase treatment (Figure 5D), telomere replication was likely not affected.Figure 4.

Bottom Line: In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species.Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution.In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres.

View Article: PubMed Central - PubMed

Affiliation: Telomeres and Cancer laboratory, 'Equipe Labellisée Ligue contre le Cancer', UMR3244, Institut Curie, 26 rue d'Ulm, 75248 Paris, France.

ABSTRACT
In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species. Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution. Here, we show that telomeres on chromosome arms carrying subtelomeric heterochromatic caps in the chimpanzee, which are completely absent in humans, replicate later than telomeres on chromosome arms without caps. In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres. Strikingly, telomere-containing RNA accumulates extrachromosomally in gorilla mitotic cells, suggesting that at least some aspects of telomere-containing RNA biogenesis have diverged in gorilla, perhaps in concert with the evolution of heterochromatic caps in this species.

Show MeSH
Related in: MedlinePlus