Quantitative Fitness Analysis Shows That NMD Proteins and Many Other Protein Complexes Suppress or Enhance Distinct Telomere Cap Defects

Abstract

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.

A Naturally Thermolabile Activity Compromises Genetic Analysis of Telomere Function in Saccharomyces cerevisiae

“…This point is further illustrated by the complex epistatic interaction that we observed between cgi121-Δ and cdc13-Δ mutations at different temperatures ( Figure 8). These results also have implications for genome-wide suppression and enhancer screens that have monitored viability of cdc13-1 and yku70-Δ strains at temperatures ranging from 36°to 37.5° (Downey et al 2006;Addinall et al 2008Addinall et al , 2011. At least some subset of genes recovered from these screens is presumably due to a genetic interaction with the Tmp 2 phenotype, rather than with either CDC13 or YKU70.…”

Sphingolipids regulate telomere clustering by affecting transcriptional levels of genes involved in telomere homeostasis

“…S1F), suggesting that the suppression of the temperature-sensitive growth defect in arv1Δ mutant caused by overexpression of EBS1 or UPF1 is not due to restoration of their functional impairments. Because expression of genes encoding telomerase subunits and regulators, such as EST1, EST2, EST3, TEN1 and STN1, has been shown to be upregulated by loss of the NMD pathway (such as in the upf1Δ mutant) (Addinall et al, 2011;Dahlseid et al, 2003;He et al, 2003), and because Stn1 is a negative regulator of telomerase action (Addinall et al, 2011;Dahlseid et al, 2003;Puglisi et al, 2008), the suppression of phenotypes in arv1Δ mutant by overexpression of EBS1 or UPF1 can be explained by a decreased expression of STN1 through NMD. This is consistent with our observation that EBS1 overexpression rescued not only the temperature-sensitive growth defect (Fig.…”

RNA decay, evolution, and the testis

“…While it is not known whether human UPF2 has non-NMD functions, yeast UPF2 has been shown to function in telomeres and chromatin silencing. 1,12,27 Some mammalian NMD factors, including UPF1, have been shown to have non-NMD functions, including in the Staufen-mediated mRNA decay pathway. 38 Thus, it is possible that mammalian UPF2 has a non-NMD function and that disruption of this function has a role in the spermatogenic defects observed by Bao et al However, it is worth noting that studies in Drosophila, zebrafish and mice have shown that loss or depletion of several different NMD factors, including UPF2, lead to very similar defects, suggesting that perturbed NMD is responsible.…”