A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging

Abstract

Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.

Figure 1

A. Summary of RLS data for long-lived deletion strains. Axes indicate % increase in RLS relative to control in MATa and MATα respectively. Point size is proportional to number of mother cells scored, and point color indicates p-value for increased RLS relative to control. Point outline indicates stringency for inclusion: high stringency cutoff was p<0.05 for Wilcoxon rank-sum increased survival independently in both mating types, and low stringency was p<0.05 for pooled data from both mating types with increased RLS shown in each mating type alone. B. Functional clustering of long-lived deletions. Large circles represent long-lived deletions; edges are published physical protein-protein interactions. Overrepresented categories noted in color; p<0.05 with Holm-Bonferroni multiple testing correction.

Figure 2

A. Estimated false positive rate for given percent increase in RLS and number of mother cells n. x-axis indicates % increase in RLS relative to wild type control, y-axis indicates sample size n, and z-axis and point color indicate estimated false positive proportion. B. Estimated false negative rate at a threshold of 30% increased RLS, for given actual percent increased RLS and number of mother cells n. x-axis indicates actual % increase expected for the genotype, z-axis indicates the estimated false negative proportion using a 30% screening cutoff, and point color indicates sample size n (black, n=5, blue, n=10, red, n=40).

Figure 3

Regulators of nuclear tRNA import and export influence lifespan, and DR and los1Δ act in the same pathway. A. Deletion of the nuclear tRNA exporter gene LOS1 extends RLS. B. Deletion of tRNA importer gene MTR10 does not reduce RLS. C. Additional copy of the nuclear tRNA importer MTR10, which functions antagonistically to LOS1, extends RLS. D. Blinded scoring of fluorescence microscopy reveals that DR (0.05% glucose) reduces nuclear Los1-GFP, ***p<0.001. Error bars denote the standard error of the mean (s.e.m.). E through I. RLS analysis indicates that deletion of LOS1 acts similarly to DR. Deletion of LOS1 fails to extend RLS of sir2Δ cells, but robustly extends RLS of sir2Δfob1Δ cells. Deletion of LOS1 does not increase RLS additively with DR or with genetic mimics of DR (tor1Δ and sch9Δ). RLS and microscopy statistics are provided in Supplemental Table S5.

Figure 4

Rad53 acts between DR and Los1, but not Tor1, is enhanced by DR, and modulates lifespan. A. Fluorescence microscopy indicates that Rad53 is required for nuclear exclusion of Los1-GFP in response to DR. B. RLS analysis demonstrates that Rad53 is not required for RLS extension by deletion of either LOS1 or C. TOR1. D. Fluorescence microscopy indicates that Rad53 is not required for nuclear exclusion of Los1-GFP in response to deletion of TOR1 or E. by addition of 1nM rapamycin. F. Levels of Rad53-GFP in the cell, normalized to 2% glucose growth conditions, as assayed by flow cytometry. G.GFP-tagged Rad53 is enriched in the nucleus by DR. H. RLS analysis demonstrates that Rad53 is required for RLS extension from DR. I. Additional copy of RAD53 extends RLS. J. Representative growth curves of RAD53 overexpression. K. Mother cell divisions of Rad53-OE and wild type on agar as a function of chronological time. RLS and microscopy statistics are provided in Supplemental Table S5. *p<0.05, ***p<0.001. Error bars denote the standard error of the mean (s.e.m.).

Figure 5

Deletion of LOS1 activates GCN4 without reducing global mRNA translation, and Gcn4 is necessary for RLS extension by los1Δ. A. Relative proportions of Gcn4 regulated genes within the subset of genes found via microarray to be upregulated in los1Δ yeast. B. RT-qPCR verification of a subset Gcn4 target genes found to be upregulated by microarray analysis of los1Δ cells, focused on enriched GO term related genes. C. Representative growth curves, D. polysome profiles, and E. cell cycle profiles of los1Δ, tor1Δ, and WT yeast indicate that neither los1Δ nor tor1Δ cells have a detectable defect in mRNA translation. For comparison, a translation-deficient rpl20Δ mutant is shown in each case. R values in panel E correspond to the number of ribosomes bound to a particular mRNA. F. Deletion of GCN4 partially blocks RLS extension from deletion of LOS1. G. Deletion of GCN4 largely prevents RLS extension in genetic models of DR, but not the DR independent Ubr2 pathway. Percent suppression of RLS is calculated by comparing the percent mean RLS extension of the long lived mutant in wild type and gcn4Δ backgrounds. RLS statistics are provided in Supplemental Table S5. H. Diagram summarizing the comprehensive model for DR supported by the data from this study. Error bars denote the standard error of the mean (s.e.m.).