Multiomics of GCN4-Dependent Replicative Lifespan Extension Models Reveals Gcn4 as a Regulator of Protein Turnover in Yeast

Abstract

We have shown that multiple tRNA synthetase inhibitors can increase lifespan in both the nematode C. elegans and the budding yeast S. cerevisiae by acting through the conserved transcription factor Gcn4 (yeast)/ATF-4 (worms). To further understand the biology downstream from this conserved transcription factor in the yeast model system, we looked at two different yeast models known to have upregulated Gcn4 and GCN4-dependent increased replicative lifespan. These two models were rpl31aΔ yeast and yeast treated with the tRNA synthetase inhibitor borrelidin. We used both proteomic and RNAseq analysis of a block experimental design that included both of these models to identify GCN4-dependent changes in these two long-lived strains of yeast. Proteomic analysis of these yeast indicate that the long-lived yeast have increased abundances of proteins involved in amino acid biosynthesis. The RNAseq of these same yeast uncovered further regulation of protein degradation, identifying the differential expression of genes associated with autophagy and the ubiquitin-proteasome system (UPS). The data presented here further underscore the important role that GCN4 plays in the maintenance of protein homeostasis, which itself is an important hallmark of aging. In particular, the changes in autophagy and UPS-related gene expression that we have observed could also have wide-ranging implications for the understanding and treatment of diseases of aging that are associated with protein aggregation.

Keywords: ATF-4; ATF4; Gcn4; autophagy; tRNA synthetase; ubiquitin–proteasome system.

Figure 1

Gcn4/ATF-4/ATF4 and its highly conserved nature of activation. (A) Gcn4/ATF-4/ATF4 is regulated via upstream open reading frames (uORFs). (B) The uncharged tRNA sensor, namely Gcn2/GCN-2/GCN2, can lead to a signal cascade and the translation of Gcn4/ATF-4/ATF4 in a highly conserved manner.

Figure 2

Proteomic analysis of long-lived yeast. (A) Block design used for the genomic analysis. (B) Volcano plot showing the results of the linear model fitted to Gcn4 translation. Red dots indicate that the gene is significantly differentially expressed from the linear model fitted to reported Gcn4 translation levels (p_adj < 0.05. Log₂ fold change > |0.01|). (C) GeneMania interaction and biological process analysis of differentially abundant proteins (p_adj < 0.01). (D) Heatmap of the differentially abundant proteins (p_adj < 0.01) from the linear model fitted to Gcn4 translation.

Figure 3

RNA sequencing of long-lived yeast. (A) Principal component (PC) analysis of the sequenced and analyzed samples. (B) Volcano plot showing the result of the linear model fitted to Gcn4 translation. Red dots indicate that the gene is significantly differentially expressed from the linear model fitted to reported Gcn4 translation levels (p_adj < 1 × 10⁻⁴, Log₂ fold change > |0.1|). (C) GeneMania interaction and biological process analysis of these differentially expressed genes (p_adj < 1 × 10⁻⁴). (D) Heatmap showing the mRNA abundance of the 27 genes found to be differentially expressed from the linear model fitted to Gcn4 translation (p_adj < 1 × 10⁻⁴).