During the fellowship we have been able to carry out excellent work and publishing it in Cell journal. To start with, we have been able to generate Rtel1f/f MEFs expressing either Rtel1 WT or the different HHS mutants at close to physiological levels. This have allowed us to study the phenotype of these cells after Rtel1 deletion. We have analysed the different kind of telomeric dysfunction that these cells present, including telomeric loss, telomeric fragility and telomeric heterogeneity. We have also performed quantitative FISH in these different mutants to check telomeric size. On a second phase of the project, we cloned all the different mutants and transduced 293 cells with a control vector, WT Rtel1 or the different Rtel1 mutants. These cells express Rtel1 at different extends between them. We have performed SILAC experiments to identify new or lost interaction partners of Rtel1 in the different mutants. This analysis was a bit difficult to perform due to the different expression levels of the different mutants. We will have to analyse the data from 2 different SILAC experiments and see if other cellular systems would allow for better expression levels between the different mutants. However, this part of project has been put on stand by due to the surprising observation that Terc deficient cells rescue the telomeric dysfunction observed in Rtel1 deficient cells. We have been able to gain insight into this mechanism by different experiments.
Terc is the RNA component of telomerase, an enzyme that solves the end replication problem by extending telomere repeats, being therefore essential for stem cell renewal and tissue homeostasis. However, telomerase is a “double-edged sword” as its re-expression in ∼90% of all human cancers is sufficient to drive transformation and provide unlimited proliferative capacity. We made the unexpected discovery that telomerase is also the driver of telomere catastrophe in Rtel1-deficient cells. We establish that this pathological effect of telomerase results from its aberrant binding and stabilization of reversed replication forks within the telomere, which inhibits telomere replication. Once bound by telomerase, the only option to resolve the stalled replication fork is to recruit SLX1/4 to excise the offending DNA secondary structure, which results in dramatic consequences for the telomere. Our conclusion that telomerase binds inappropriately to reversed replication forks that form within telomeres in Rtel1-null cells is supported by PLA and RNAscope experiments, which revealed that telomerase binds aberrantly to telomeres in these cells. PLA for TERT-RAD51 also placed telomerase in close proximity to reversed replication forks in the absence of RTEL1. Furthermore, blocking fork reversal abolished aberrant telomerase binding to telomeres in Rtel1-null cells. In turn, this prevented the accumulation of SLX1/4 at telomeres, which catalyzes t-loop excision, overcame the toxic effect of telomerase in Rtel1−/− cells, and suppressed telomere dysfunction. We propose that blocking fork reversal prevents the fork from slowing and allows the replisome to replicate unimpeded through the telomere displacing the t-loop in its wake. The importance of fork reversal for this phenomenon is also supported by our analysis of fork restart activities in the context of Rtel1 and telomerase deficiency. Inhibition of fork restart mimics the pathological effect of telomerase and is sufficient to induce telomere dysfunction in Rtel1−/−Tert−/−cells. We hypothesized that binding of telomerase to the reversed fork would prevent efficient replication restart, possibly by outcompeting fork restart activities. In agreement with this possibility, analysis of replication dynamics at telomeres by SMARD demonstrated that telomerase inhibits active telomere replication in cells lacking RTEL1, whereas removing telomerase mitigates this effect. These results establish that telomerase binding to reversed replication forks is inhibitory for replication fork restart within telomeres. This work was published in the prestigious Cell journal (Margalef et al., 2018).
