Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast

Abstract

The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1–PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1–PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

The Role of Rif1 in telomere length regulation is separable from its role in origin firing

“…Our results differ from those of Kedziora et al, who conclude that PP1 binding to Rif1 regulates both origin firing and telomere length (Kedziora et al, 2018). This group made mutations in four regions of Rif1 predicted to bind PP1, while our study and the study by Mattarocci mutated only Figure 4 A…”

“…We passaged the rif1-pp1bs mutants in liquid culture to overcome any phenotypic delay in telomere lengthening and still found no change in telomere length compared to WT, whereas rif1∆ continued to elongate ( Figure 1F). This result differs from a previous study, which found that telomeres were longer in a different RIF1 mutant that disrupted PP1 binding (Kedziora et al, 2018). To further probe this discrepancy, we examined additional RIF1 PP1 binding mutants reported in the literature (Supplemental Figure 2A relating to Figure 1).…”

“…-5 +5 0 two sites (Mattarocci et al, 2014). To address the discrepancy in our conclusions, we mutated the additional sites 3 and 4 as described by (Kedziora et al, 2018), and found that this 4 site mutant rif1-pp1bs-4 showed very low protein levels on a western blot (Supplemental Figure 2 relating to Figure 1) suggesting the additional site 3 and 4 mutations destabilized the protein.…”

“…We also found that Mcm4 phosphorylation levels increased in rif1-pp1bs and rif1-R/S to a similar extent (Supplemental Figure 2D,E relating to Figure 1). In contrast, the RIF1 PP1 binding mutant reported in (Hiraga et al, 2014) and further analyzed in (Kedziora et al, 2018), rif1-pp1bsD, in which two additional putative PP1 binding sites 3 and 4 were mutated, was not stably expressed (Supplemental Figure 2A,F relating to Figure 1). To further examine the potential stability issues, we mutated the additional sites 3 and 4 in our construct to generate rif1-pp1bs-4 and found this protein was also not stably expressed (Supplemental Figure 2A,G relating to Figure 1).…”

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks

“…Rif1 associates with telomeric regions, consistent with its function in controlling telomere length and replication timing near telomeres . Rif1 additionally binds the HML , HMR and MAT loci, interacting with Rap1 at these sites as at telomeres .…”