CAN1 Arginine Permease Deficiency Extends Yeast Replicative Lifespan via Translational Activation of Stress Response Genes

Abstract

Transcriptional regulation plays an important role in the control of gene expression during aging. However, translation efficiency likely plays an equally important role in determining protein abundance, but it has been relatively understudied in this context. Here, we used RNA sequencing (RNA-seq) and ribosome profiling to investigate the role of translational regulation in lifespan extension by CAN1 gene deletion in yeast. Through comparison of the transcriptional and translational changes in cells lacking CAN1 with other long-lived mutants, we were able to identify critical regulatory factors, including transcription factors and mRNA-binding proteins, that coordinate transcriptional and translational responses. Together, our data support a model in which deletion of CAN1 extends replicative lifespan through increased translation of proteins that facilitate cellular response to stress. This study extends our understanding of the importance of translational control in regulating stress resistance and longevity.

Keywords: Saccharomyces cerevisiae, mRNA-binding proteins; aging; amino acid transport; post-transcriptional gene regulation; ribosome profiling.

Figure 1. Deletion of CAN1 Increases Replicative Lifespan in Yeast

(A) Survival curves for can1Δ and corresponding experiment-matched wild-type cells. Data obtained for the MATa and MATα deletion strains are pooled. Mean lifespans and the number of cells assayed are shown in parentheses. (B) Transcriptional and translational changes in the can1Δ mutant. Significantly up-regulated and down-regulated genes in can1Δ are grouped in accordance to whether they are affected by a change in mRNA transcription (as quantified by RNA-seq), translation efficiency or by a combined effect. (C) Pathway enrichment analysis. Top hits down-regulated or up-regulated in can1Δ cells were analyzed using DAVID.

Figure 2. Lifespan Extension in can1Δ Mutant is Dependent on Gcn4 and Hac1 Transcription Factors, and Deletion of CAN1 Increases Lifespan Additively with DR

(A) Deletion of CAN1 translationally activates Gcn4 through upstream translated uORFs. (B) GCN4 footprint levels (rpkm) in wild-type and can1Δ cells. Results are represented as means ± SEM from three independent experiments. (C) GCN4-luciferase translation is increased in can1Δ cells similar to the levels induced by argi-nine starvation in wild-type cells (-ARG). Results are represented as means ± SEM from three independent experiments, *p<0.05. (D) 76% of genes up-regulated in can1Δ mutant are annotated as Gcn4 targets on YEASTRACT. (E) The up-regulation of Gcn4 target genes in can1Δ cells was verified by qPCR. Results are represented as means ± SEM from three independent experiments; *p<0.05, ***p<0.001. The sequences of the primers used for qPCR are listed in Table S4, related to Figure 2. (F) Replicative lifespan in can1Δ mutant is further extended (p < 0.05) in response to DR by reducing the concentration of glucose in the media from 2% to 0.05%. (G) The up-regulation of Hac1 target genes in can1Δ cells was verified by qPCR. Results are represented as means ± SEM from three independent experiments; **p<0.01, ***p<0.001. (H) Extended lifespan in can1Δ is dependent on the Hac1 transcription factor. Mean lifespans and the number of cells assayed are shown in parentheses.

Figure 3. Deletion of CAN1 Promotes ER Stress Resistance and Activates Gcn4 without Reducing Global mRNA Translation

(A) Genes translationally up- or down-regulated in can1Δ only partially overlap with rpl22aΔ and tor1Δ. (B) Cells lacking CAN1 are resistant to tunicamycin. 10× serial dilutions of logarithmically growing cells were spotted on agar plates with indicated concentrations of tunicamycin and incubated for 48 h at 30°C. (C–D) Polysome profiles of can1Δ, tor1Δ and rpl22aΔ mutants and cells under DR conditions indicate that neither CAN1 nor TOR1 deletion leads to inhibition of global mRNA translation. Representative polysome profiles (C) from three independent experiments are shown, and the area under polysome peaks was quantified using ImageJ software (D); **p<0.01. Error bars denote the standard error of the mean (SEM). (E) Representative growth curves of can1Δ, tor1Δ and rpl22aΔ mutants. The doubling time for each strain is provided in Figure S2F, related to Figure 3.

Figure 4. Translational Regulation of Gene Expression in the Long-Lived Mutants

(A–B) Genes, whose TE was increased (A) or decreased (B) more than 1.5-fold in can1Δ, were visualized using STRING. (C) Protein-protein interaction network of the proteins whose TE was changed more than 1.5-fold in tor1Δ. Genes whose TE was increased are indicated in yellow, and genes whose TE was decreased are shown in blue. Up-regulated mRNA-binding proteins are highlighted in red. (D) Deletion of TOR1 confers increased resistance to Cu.

Figure 5. Translational Control by mRNA-Binding Proteins is Closely Interlinked with the Regulation of Gene Expression by Transcription Factors

(A) Heatmap displaying transcription factors and mRNA-binding proteins that show significant change in the long-lived mutants either at transcriptional or translational levels. Changes in log2 mRNA and footprints rpkm, and log2 TE change are shown. (B) The regulatory network showing interactions between transcriptional and translational regulators in the long-lived mutants. Genetic alterations (CAN1, TOR1, RPL22A) are represented as orange triangles. Aging-associated transcription factors (TFs) and mRNA-binding proteins (RBPs) are shown as blue and red nodes respectively. (C) A model for the role of post-transcriptional gene regulation in aging. Post-transcriptional control by RBPs is closely interlinked with the regulation of transcription by TFs allowing coordinated changes in protein translation.