Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in Saccharomyces cerevisiae
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Evaluation Summary:
This study investigates the tolerance to change in the sequence of telomere repeats, by analyzing a strain expressing mutant TLC1, the RNA component of the telomerase. The authors conclude that Rif2 protects telomere ends in the absence of RAP1 by inhibiting MRX and promoting Rad51-dependent homologous recombination to maintain telomere homeostasis. The study clarifies the role of Rif2 in telomere homeostais and describes how cells can extend telomeres and control senescence in the absence of Rap1 binding to telomeres. The possibility of coping with telomere sequence modification through flexibility and redundancy of capping proteins is of general interest in terms of telomere evolution.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Rap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae . Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. Rif2 and the Ku complex work in parallel to prevent tlc1-tm telomere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.
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Evaluation Summary:
This study investigates the tolerance to change in the sequence of telomere repeats, by analyzing a strain expressing mutant TLC1, the RNA component of the telomerase. The authors conclude that Rif2 protects telomere ends in the absence of RAP1 by inhibiting MRX and promoting Rad51-dependent homologous recombination to maintain telomere homeostasis. The study clarifies the role of Rif2 in telomere homeostais and describes how cells can extend telomeres and control senescence in the absence of Rap1 binding to telomeres. The possibility of coping with telomere sequence modification through flexibility and redundancy of capping proteins is of general interest in terms of telomere evolution.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
This study investigates the tolerance to change in the sequence of telomere repeats, by analyzing a strain expressing mutant TLC1, the RNA component of the telomerase. The authors conclude that Rif2 protects telomere ends in the absence of RAP1 by inhibiting MRX and promoting Rad51-dependent homologous recombination to maintain telomere homeostasis. The study clarifies the role of Rif2 in telomere homeostais and describes how cells can extend telomeres and control senescence in the absence of Rap1 binding to telomeres. The possibility of coping with telomere sequence modification through flexibility and redundancy of capping proteins is of general interest in terms of telomere evolution.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This is a nicely and well-performed study on the role of Rap1 and Rif2 in telomere homeostasis of the yeast Saccharomyces. Using a tlc1 mutant that generates telomere repeats with a lower Rap1 binding plus additional mutations, the authors show that Rap1 binding to telomeres is not necessary for cell viability and causes lengthening of telomeres. The authors show that the viability produced without Rap1 binding is not due to Tfb1 action, as it could have been predicted from previous studies. Analysis of the need for Rif1 or Rif2, which when bound to Rap1 inhibits telomerase, the authors find that it is Rif2 and not Rif1 the essential counterpart. The authors show indeed that indeed Rif1 and Sir4 binding to telomeres tlc1-tm is decreased but not that of Rif2 and Sir3. The authors focus the manuscript on the …
Reviewer #1 (Public Review):
This is a nicely and well-performed study on the role of Rap1 and Rif2 in telomere homeostasis of the yeast Saccharomyces. Using a tlc1 mutant that generates telomere repeats with a lower Rap1 binding plus additional mutations, the authors show that Rap1 binding to telomeres is not necessary for cell viability and causes lengthening of telomeres. The authors show that the viability produced without Rap1 binding is not due to Tfb1 action, as it could have been predicted from previous studies. Analysis of the need for Rif1 or Rif2, which when bound to Rap1 inhibits telomerase, the authors find that it is Rif2 and not Rif1 the essential counterpart. The authors show indeed that indeed Rif1 and Sir4 binding to telomeres tlc1-tm is decreased but not that of Rif2 and Sir3. The authors focus the manuscript on the role of Rif2 which was known to bind dsDNA and MRX to show that Rif2 protects telomere ends in the absence of RAP1 by inhibiting MRX and promoting Rad51-dependent homologous recombination. Further analysis allows the authors to conclude that Rif2 and the Yku complex protects such telomeres. The study is well-executed with the appropriate molecular analysis of telomeres, protein binding by ChIP and senescence studies using a clever and well-explained rational combination of mutants, but the authors need to emphasize more the novelty of their conclusions, they should clarify and better support the conclusions about the function of Rif2 and provide more details on the role of Sir3 in tlc1-tm mutants.
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Reviewer #2 (Public Review):
This is a very interesting study addressing how telomeres can be maintained when the main budding yeast shelterin protein (Rap1) is missing. The authors used a telomerase RNA template mutant to generate telomere repeats lacking high-affinity Rap1 binding sites. Nicely, the elongation of the mutant telomeres becomes length-independent, the expected hallmark of Rap1-depleted telomeres combined with a compromised telomerase. ChIP analyses also show that mutant telomeres are to a large extent Rap1-depleted. Among the Rap1-interacting factors tested, Rif1 and Sir4 are also missing or reduced but surprisingly, not Rif2 and Sir3. Furthermore, Rif2 is now essential for telomere stability and cell viability by antagonizing the uncontrolled action of the MRX complex (mostly end resection). The authors conclude that …
Reviewer #2 (Public Review):
This is a very interesting study addressing how telomeres can be maintained when the main budding yeast shelterin protein (Rap1) is missing. The authors used a telomerase RNA template mutant to generate telomere repeats lacking high-affinity Rap1 binding sites. Nicely, the elongation of the mutant telomeres becomes length-independent, the expected hallmark of Rap1-depleted telomeres combined with a compromised telomerase. ChIP analyses also show that mutant telomeres are to a large extent Rap1-depleted. Among the Rap1-interacting factors tested, Rif1 and Sir4 are also missing or reduced but surprisingly, not Rif2 and Sir3. Furthermore, Rif2 is now essential for telomere stability and cell viability by antagonizing the uncontrolled action of the MRX complex (mostly end resection). The authors conclude that Rif2 recruitment to telomeres is in part Rap1-independent, highlighting the built-in redundancy among the pathways ensuring telomere protection.
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Reviewer #3 (Public Review):
This manuscript reports a detailed description of telomere homeostasis in a strain expressing a previously described mutant allele of TLC1, the gene encoding the RNA component of the telomerase. The tlc1-tm RNA produces telomere repeats without GGG. It is shown here that tlc1-tm telomeres present a strong defect in Rap1 binding. Nevertheless, tlc1-tm cells are viable with a growth rate similar to wild-type cells. As expected the association of Rif1 and Sir4 is decreased but not that of Cdc13, Ku and more surprisingly Rif2 and Sir3. The tlc1-tm telomeres also show increased recruitment of the MRX complex. Rif2 binding is dependent on RAD50 specifically in tlc1-tm cells. Taken together, it seems that the stability of tlc1-tm telomeres combines the classical telomere protection functions provided by Cdc13 and …
Reviewer #3 (Public Review):
This manuscript reports a detailed description of telomere homeostasis in a strain expressing a previously described mutant allele of TLC1, the gene encoding the RNA component of the telomerase. The tlc1-tm RNA produces telomere repeats without GGG. It is shown here that tlc1-tm telomeres present a strong defect in Rap1 binding. Nevertheless, tlc1-tm cells are viable with a growth rate similar to wild-type cells. As expected the association of Rif1 and Sir4 is decreased but not that of Cdc13, Ku and more surprisingly Rif2 and Sir3. The tlc1-tm telomeres also show increased recruitment of the MRX complex. Rif2 binding is dependent on RAD50 specifically in tlc1-tm cells. Taken together, it seems that the stability of tlc1-tm telomeres combines the classical telomere protection functions provided by Cdc13 and KU with features of a double-strand break with increased recruitment of MRX and therefore of Rif2 that in turn restricts MRX action.
The manuscript is well written. Nevertheless, there are two major points that should be clarified:
1. The vocabulary used to describe the decreased binding of Rap1 to tlc1-tm telomeric repeats should be standardized taking into account that 1) Rap1 binding is not totally abolished 2) telomeres (except TELVII-L mut) retain RAP1 binding sites in their most proximal part as well as on interstitial telomeric repeats in Y' telomeres. It seems therefore more correct to speak of a decreased level of Rap1 rather than loss or lack.
2. The role of recombination in the homeostasis of tlc1-tm telomeres should be discussed because stochastic recombination events or processing of intermediates could be at the origin of variations of the telomere profiles between isogenic tlc1-tm clones. In this line, the figure 5-fig Supp2 shows a clear difference in the telomere profiles of tlc1-tm and tlc1-tm rad52 cells but this is not commented in the text. Similarly, the large heterogeneity of X-telomeres is not mentioned though it could be due to de novo acquisition of Y' sequences at least in some cases. Finally, Rad52 is required for the growth of rif2 tlc1-tm cells. -
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