Numbers of known aging associated genes (AAGs) reported in the scientific literature to affect (increase/decrease CLS/RLS) under a variety of conditions

The gene lists were extracted, after processing, from downloaded files (as of 8th November 2022) using the databases SGD 22 and GenAge 23. The actual gene lists are available in Additional File 1’. The signs ‘+’ and ‘-‘ indicate that the respective annotation property is present or missing in that group of genes. We present the total number of genes in each category as the number of, as a trend, severely understudied/uncharacterized genes among those. The columns ‘RUG’ and ‘RDG’ show the numbers of rapamycin-up– and –downregulated genes in each of the 15 categories.

YBR238C deletion increases the cellular lifespan

(A and B) Expression analysis of YBR238C gene by qRT-PCR in yeast Saccharomyces cerevisiae genetic backgrounds BY4743 (A) and CEN.PK113-7D (B). Data are represented as means ± SD (n=4). ****P < 0.0001 based on two-sided Student’s t-test.

(C and D) Chronological lifespan (CLS) of the wild type and ybr238cΔ mutant was assessed in the SD medium supplemented with auxotrophic amino acids for BY4743 (C) and only the SD medium for CEN.PK113-7D (D) strains in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 2. Data are represented as means ± SD (n=3) (C) and (n=4) (D). **P < 0.01, ***P < 0.001, and ****P < 0.0001 based on two-way ANOVA followed by Šídák’s multiple comparisons test.

(E and F) CLS of the wild type and ybr238cΔ mutant was performed in the flask. Outgrowth was performed for ten-fold serial diluted aged cells onto the YPD agar plate (E) and YPD medium in the 96-well plate (F). The serial outgrowth of aged cells on agar medium was imaged (E) and quantified the survival relative to outgrowth of day 2 (F). A representative of two experiments for (E) and (F) is shown.

Longevity signatures of ybr238cΔ mutant

(A and B) Functional enrichment analysis of upregulated genes in yeast Saccharomyces cerevisiae ybr238cΔ mutant compared to wild type (BY4743). Bar plots showing the enriched biological process (A) and identified MCODE complexes based on the top-three ontology enriched terms (B). See also Additional File 3.

(C and D) ATP analysis of wild type and ybr238cΔ mutant for BY4743 (C) and CEN.PK113– 7D (D). Data are represented as means ± SD (n=6). **P < 0.01, and ****P < 0.0001 based on two-sided Student’s t-test.

(E and F) Expression analysis of MSN4 gene by qRT-PCR in yeast Saccharomyces cerevisiae genetic backgrounds BY4743 (E) and CEN.PK113-7D (F). Data are represented as means ± SD (n=2). **P < 0.01 based on two-sided Student’s t-test.

(G and H) ROS analysis of wild type and ybr238cΔ mutant for BY4743 (G) and CEN.PK113– 7D (H). Data are represented as means ± SD (n=4). ***P < 0.001, and ****P < 0.0001 based on two-sided Student’s t-test.

YBR238C affects cellular aging via HAP4-dependent and independent mechanisms

(A) The HAP4 regulon within the upregulated DEGs of ybr238cΔ mutant. See also Additional File 3.

(B and C) Expression analysis of HAP4 gene by qRT-PCR in yeast Saccharomyces cerevisiae genetic backgrounds BY4743 (B) and CEN.PK113-7D (C). Data are represented as means ± SD (n=2). **P < 0.01 based on two-sided Student’s t-test.

(D) Heatmap of mitochondrial ETC genes expression analysis by qRT-PCR in CEN.PK113– 7D strains. Data are represented as means ± SD (n=2).

(E, F and H) Chronological lifespan (CLS) of yeast Saccharomyces cerevisiae genetic background CEN.PK113-7D strains was performed in the SD medium in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 2. Data are represented as means ± SD (n=6). *P < 0.05, ***P < 0.001, ****P < 0.0001 and ns: non-significant. Ordinary one–way ANOVA followed by Tukey’s multiple comparisons test (E and F). Two-way ANOVA followed by Šídák’s multiple comparisons test (H).

(G) Expression analysis of HAP4 and ETC genes by qRT-PCR in YBR238C deletion (ybr238cΔ) and overexpression (YBR238C-OE) strains of yeast Saccharomyces cerevisiae genetic background CEN.PK113-7D (C). Data are represented as means ± SD (n=2). **P < 0.01, ***P < 0.001 and ****P < 0.000 based on Two-way ANOVA followed by Šídák’s multiple comparisons test.

(I) Model representing regulation of cellular aging by YBR238C via HAP4–dependent and independent mechanisms.

RMD9 deletion decreases the cellular lifespan

(A and E) Chronological lifespan (CLS) of yeast Saccharomyces cerevisiae genetic background CEN.PK113-7D strains was performed in the SD medium in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 2. Data are represented as means ± SD (n=12) (A) and (n=6) (B). ****P < 0.0001 and ns: non-significant. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (A). Two-way ANOVA followed by Dunnett’s multiple comparisons test (E).

(B, C, F and G) CLS of yeast strains was performed in the flask. Outgrowth was performed for ten-fold serial diluted aged cells onto the YPD agar plate (B and F) and YPD medium in the 96-well plate (C and G). The serial outgrowth of aged cells on agar medium was imaged (B and F) and quantified the survival relative to outgrowth of day 1 (C and G). A representative of two experiments for B, C, F and G is shown.

(D) Serial dilution growth assays of the yeast strains on fermentative glucose medium (YPD) and respiratory glycerol medium (YPG).

(H) ATP level in yeast strains. Data are represented as means ± SD (n=3). *P < 0.05, and ***P < 0.001 based on ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. See also Figure S5.

YBR238C affects cellular aging via RMD9– dependent mechanism

(A, B, G and H) Chronological lifespan (CLS) of yeast Saccharomyces cerevisiae genetic background CEN.PK113-7D strains was performed in the SD medium in the flask. Outgrowth was performed for ten-fold serial diluted aged cells onto the YPD agar plate (A and G) and YPD medium in the 96-well plate (B and H). The serial outgrowth of aged cells on agar medium was imaged (A and G) and quantified the survival relative to outgrowth of day 2 (B and H). A representative of two experiments for A, B, G and H is shown.

(C and I) CLS of yeast strains was performed in the SD medium in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 2. Data are represented as means ± SD (n=6) (C) and (n=12) (I). ****P < 0.0001 based on two-way ANOVA followed by Tukey’s multiple comparisons test (C) and Dunnett’s multiple comparisons test (I). ns: non-significant.

(D and J) ATP level in yeast strains. Data are represented as means ± SD (n=4). *P < 0.05, ***P < 0.001, and ****P < 0.0001 based on on ordinary one-way ANOVA followed by Tukey’s multiple comparisons test.

(E and K) ROS level in yeast strains. Data are represented as means ± SD (n=4). **P < 0.01, ***P < 0.001, and ****P < 0.0001 based on ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. ns: non-significant.

(F) Expression analysis of RMD9 gene by qRT-PCR in yeast strains. Data are represented as means ± SD (n=4). ***P < 0.001, and ****P < 0.0001 based on ordinary one-way ANOVA followed by Tukey’s multiple comparisons test.

(L) Effect of antimycin A (AMA) treatment on CLS of yeast strain was assessed in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 1. A representative heatmap data of three experiments is shown.

(M) Model representing regulation of cellular aging by YBR238C via HAP4 and RMD9 dependent mechanisms.

TORC1-MItochondria-TORC1 (TOMITO) signaling axis regulate cellular aging

(A) ATP analysis of logarithmic phase wild type CEN.PK113-7D yeast cells treated with rapamycin for 1 hour. Data are represented as means ± SD (n=3). ****P < 0.0001 based on ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. See also Figure S5C.

(B) Expression analysis of mitochondrial ETC genes by qRT-PCR. Data are represented as means ± SD (n=2).

(C-G) Chronological lifespan (CLS) of yeast strains with indicated concentrations of rapamycin was performed in the SD medium in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 2. Data are represented as means ± SD (n=3). ***P < 0.001, and ****P < 0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons test. ns: non-significant.

(H) CLS of wild type yeast strain with indicated concentrations of rapamycin and antimycin A (10 µM) was performed in the SD medium in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 1. Data are represented as means ± SD (n=3). ****P < 0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons test. ns: non– significant. ns: non-significant.

(I) ATP analysis of stationary phase wild type CEN.PK113-7D yeast cells were incubated with rapamycin (10 nM) and antimycin A (10 µM). Data are represented as means ± SD (n=6). ****P < 0.0001 based on ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. See also Figure S6H.

(J) Determination of human HEK293 cells survival incubated with indicated concentrations of antimycin A for 6 days. Untreated control is considered 100% cell survial. Data are represented as means ± SD (n=6).

(K) Heatmap for cell survival analysis of human HEK293 cells incubated with indicated concentrations of antimycin A and rapamycin for 6 days. Cell viability at antimycin A and rapamycin combination concentrations was measured relative to their individual treated concentration. A representative data of two experiments is shown.

Transcriptomics analysis of rapamycin to identify the TORC1 regulated genes

(A and B) Chronological lifespan (CLS) of the wild type BY4743 (A) and CEN.PK113-7D (B) yeast strains was assessed in the SD medium supplemented with auxotrophic amino acids (A) and only the SD medium (B) in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 2. Data are represented as means ± SD (n=4). *P < 0.05, and ****P < 0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons test. ns: non– significant.

(C) Principal component analysis (PCA) of the RNA-seq data from replicates of DMSO and rapamycin (50 nM) treated yeast cells (BY4743). PC1 represents 95.98% of the total variance in the experimental data, and PC2 represents 2.59% of the total variance.

(D and E) Volcano plots produced by EdgeR (D) and DESeq2 (E) demonstrate the gene expression pattern and the differentially expressed genes (DEGs) with p-value <0.05, with a fold change of log2(fold change) >1 for upregulated genes and log2(fold change) <−1 for downregulated genes.

(F) Overlap of identified DEGs using EdgeR and DESeq2 with the criteria for all two tools that p-value <0.05, with a fold change of log2(fold change) >1 for upregulated genes and log2(fold change) <−1 for downregulated genes. Upregulated and downregulated DEGs overlaps for both EdgeR and DESeq2 were further considered for functional GO enrichment analysis.

See also Additional File 2.

RNA sequencing data validation by qRT-PCR

(A and B) Expression analysis of selected genes of RNA sequencing (RNA-seq) data by qRT–PCR in in Saccharomyces cerevisiae genetic backgrounds BY4743 (A) and CEN.PK113-7D (B) wild type cells treated with DMSO and rapamycin (50 nM). Gene expression of rapamycin treated samples were compared with DMSO control. Data are represented as means ± SD (n=4). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 based on two-sided Student’s t-tests.

Identification of the role of uncharacterized YBR238C gene in cellular aging, Related to Figure 2

(A) Principal component analysis (PCA) of the RNA-seq data from replicates of wild type BY4743 and ybr238cΔ yeast strains. PC1 represents 71.75% of the total variance in the experimental data, and PC2 represents 18.15% of the total variance.

(B) Volcano plots produced by DESeq2 demonstrate the gene expression pattern and the differentially expressed genes (DEGs) with p-value <0.05, with a fold change of log2(fold change) >0.5 for upregulated genes and log2(fold change) <−0.5 for downregulated genes. GO enrichment analysis was performed for upregulated DEGs. See also Additional File 3.

(C and D) Comparison of functional enrichment analysis for upregulated DEGs of ybr238cΔ mutant and rapamycin (50 nM) treated wild type BY4743 cells. Bar plots showing the common enriched biological process (C) and MCODE complexes based on the top-three ontology enriched terms (D).

Transcription factors analysis for upregulated DEGs of ybr238cΔ mutant, Related to Figure 2

(A) The whole regulatory network of overrepresented top eight enriched transcription factors (blue) within the upregulated DEGs of ybr238cΔ mutant. Msn4 and Hap4 TFs are appearing as responsible for the highest number of regulated genes in ybr238cΔ mutant.

(B) The Msn4 regulon within the upregulated DEGs of ybr238cΔ mutant.

See also Additional File 3.

YBR238C homolog RMD9 deletion leads to mitochondrial dysfunction associated with accelerated cellular aging, Related to Figure 4

(A and E) Chronological lifespan (CLS) of yeast wild type BY4743 and deletion strains was performed in the SD medium supplemented with auxotrophic amino acids in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 1. (A) Data are represented as means ± SD (n=12). ****P < 0.0001 based on two-way ANOVA followed by Tukey’s multiple comparisons test. (E) Data are represented as means ± SD (n=4). ****P < 0.0001 based on ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. ns: non-significant.

(B, C, F and G) CLS of yeast wild type BY4743 and deletion strains was performed in the flask. Outgrowth was performed for ten-fold serial diluted aged cells onto the YPD agar plate (B and F) and YPD medium in the 96-well plate (C and G). The serial outgrowth of aged cells on agar medium was imaged (B and F) and quantified the survival relative to outgrowth of day 1 (C and G). A representative of two experiments for B, C, F and G is shown.

(D) Serial dilution growth assays of the yeast strains on fermentative glucose medium (YPD) and respiratory glycerol medium (YPG).

(H) ROS level of yeast wild type BY4743 and deletion strains. Data are represented as means ± SD (n=3). **P < 0.01, and ****P < 0.0001 based on ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.

TORC1-Mitochondrial function signaling pathways control cellular aging

(A and C) ATP analysis of wild type CEN.PK113-7D yeast cells treated with (A) antimycin A (stationary phase culture) and (C) rapamycin (logarithmic phase culture). Data are represented as means ± SD (n=3). ***P < 0.001, and ****P < 0.0001 based on ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.

(B) Chronological lifespan (CLS) of wild type yeast strain with indicated concentrations of antimycin A was performed in the SD medium in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 1. Data are represented as means ± SD (n=8). ***P < 0.001, and ****P < 0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons test. ns: non-significant. ns: non-significant.

(D and E) Expression analysis by qRT-PCR for YBR238C in logarithmic phase yeast strains treated with rapamycin (200 nM) for 1 hour. Data are represented as means ± SD (n=2). **P < 0.01, and ****P < 0.0001 based on two-sided Student’s t-test.

(F) CLS of yeast strains with indicated concentrations of rapamycin was performed in the SD medium in 96-well plate. Aged cells survival was measured relative to the outgrowth of day 2. Data are represented as means ± SD (n=3). ****P < 0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons test. ns: non-significant. See also Figure 6D.

(G) Growth assay of yeast strains with rapamycin (40 nM) for 24 and 48h. Growth was normalized with untreated control.

(H) Growth assay of yeast strains with indicated concentrations of rapamycin for 48, 72 and 96 h. Growth was normalized with untreated control.

(I) ATP analysis of stationary phase wild type CEN.PK113-7D yeast cells were incubated with indicated concentrations of rapamycin. Data are represented as means ± SD (n=6). ****P < 0.0001 based on ordinary one-way ANOVA followed by Tukey’s multiple comparisons test.

(J) ATP analysis of human HEK293 cells treated with antimycin A (10 µM) and rapamycin (100 nM) for 48 hours. Data are represented as means ± SD (n=3). ****P < 0.0001 based on ordinary one-way ANOVA followed by Tukey’s multiple comparisons test.