Yeast life span extension by depletion of 60s ribosomal subunits is mediated by Gcn4

Abstract

In nearly every organism studied, reduced caloric intake extends life span. In yeast, span extension from dietary restriction is thought to be mediated by the highly conserved, nutrient-responsive target of rapamycin (TOR), protein kinase A (PKA), and Sch9 kinases. These kinases coordinately regulate various cellular processes including stress responses, protein turnover, cell growth, and ribosome biogenesis. Here we show that a specific reduction of 60S ribosomal subunit levels slows aging in yeast. Deletion of genes encoding 60S subunit proteins or processing factors or treatment with a small molecule, which all inhibit 60S subunit biogenesis, are each sufficient to significantly increase replicative life span. One mechanism by which reduced 60S subunit levels leads to life span extension is through induction of Gcn4, a nutrient-responsive transcription factor. Genetic epistasis analyses suggest that dietary restriction, reduced 60S subunit abundance, and Gcn4 activation extend yeast life span by similar mechanisms.

Figure 1. Genome-wide screen of RP gene deletion strains verifies 14 significantly long-lived strains, each lacking an RPL gene

A–C. Survival curves for RP deletion strains that are significantly (p < 0.05) long-lived in both the MATα and MATa ORF deletion collections. Data from MATa and MATα deletion strains are pooled and experiment-matched wild-type cells are shown. Mean lifespans are shown in parentheses. (See also Table S1.)

Figure 2. Abundance of 60S ribosomal subunits correlates with RLS

A–B. Survival curves for RP paralog gene deletions and experiment-matched wild-type cells. Mean lifespans are shown in parentheses. C. Molar ratios of RP paralog transcripts (A/B). (See also Table S2). D–E. Polysome profiles of rplΔ paralogs. Long-lived deletion strains rpl31aΔ or rpl20bΔ show a reduced level of 60S ribosomal subunits relative to its paralog deletion strain (not long-lived). F. Generation time of RPL (red diamonds) and RPS (blue triangles) gene deletion strains relative to wild-type plotted versus the percent change in mean RLS relative to experiment-matched wild-type cells. Linear regressions for RPL (red) and RPS (blue) gene deletions are shown separately.

Figure 3. Interventions which decrease 60S ribosomal subunits extend RLS

A. Relative to (i) wild-type, polysome profiles for (ii) nop12Δ, (iii) ssf1Δ, (iv) rei1Δ, and (v) diazaborine-treated (15 μg/ml) cells show significant reduction of 60S subunit levels while (vi) cycloheximide-treated (25 ng/ml) cells do not. B. Deletion of 60S-specific processing factor genes NOP12, SSF1, or LOC1 increases lifespan relative to experiment-matched wild-type cells. C. Treatment of wild-type cells with 15 μg/ml diazaborine extends lifespan relative to experiment-matched wild-type cells while treatment with 25 ng/ml cycloheximide does not. Mean lifespans are shown in parentheses.

Figure 4. Depletion of 60S subunits extends RLS by a mechanism independent of Sir2 and similar to DR

A. SIR2-overexpression and fob1Δ cells show polysome profiles similar to that of wild-type. B–C. Deletion of RPL31A or diazaborine treatment (15 μg/ml) increases the RLS of sir2Δ fob1Δ cells. D. A genetic model of DR (sch9Δ) results in cells with reduced levels of both 40S and 60S ribosomal subunits and polysomes relative to wild-type. E–F. DR does not further extend the RLS of rpl31aΔ cells or cells treated with diazaborine (15 μg/ml). Mean lifespans are shown in parentheses.

Figure 5. Cells lacking RPL genes require GCN4, but not GCN2, for increased longevity

A. Gcn4-luciferease levels for rpl31aΔ, rpl31bΔ, rpl20aΔ, and rpl20bΔ relative to wild-type cells show that translation of GCN4-luciferase RNA correlates with long lifespan. Red bars represent long-lived strains and blue bars represent strains that are not long-lived. B–C. Long-lived strains rpl20bΔ and rpl31aΔ require GCN4 for full lifespan extension. D. GCN2 is not required for lifespan extension by deletion of RPL20B. Mean lifespans are shown in parentheses.

Figure 6. GCN4 is required for full lifespan extension by depletion of 60S subunits or by DR

A. Mean lifespan extension by deletion of any of 11 different RPL genes is largely dependent on GCN4 (p = <0.001). Solid bars represent the percent change in mean RLS for each rplΔ strain, relative to experiment-matched wild-type cells; hashed bars represent the percent change in mean RLS for each corresponding rplΔ gcn4Δ double mutant, relative to experiment-matched gcn4Δ cells. B. Full lifespan extension by tor1Δ (p = .03), sch9Δ (p = .21), or DR (p = .44) is dependent on GCN4. Solid bars represent the percent change in mean RLS from tor1Δ, sch9Δ or DR, relative to experiment-matched wild-type cells; hashed bars represent the percent change in mean RLS from tor1Δ, sch9Δ or DR in a gcn4Δ background, relative to experiment-matched gcn4Δ cells.

Figure 7. A genetic model for lifespan extension by DR

Lifespan extension by depletion of 60S subunits, tor1Δ, sch9Δ, or DR, is mediated in part by Gcn4; however, a portion of the lifespan extension in each case can occur via at least one Gcn4-independent mechanism.