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. 2020 Feb;19(2):e13068.
doi: 10.1111/acel.13068. Epub 2019 Dec 12.

Maf1-dependent Transcriptional Regulation of tRNAs Prevents Genomic Instability and Is Associated With Extended Lifespan

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Free PMC article

Maf1-dependent Transcriptional Regulation of tRNAs Prevents Genomic Instability and Is Associated With Extended Lifespan

Mihir Shetty et al. Aging Cell. .
Free PMC article

Abstract

Maf1 is the master repressor of RNA polymerase III responsible for transcription of tRNAs and 5S rRNAs. Maf1 is negatively regulated via phosphorylation by the mTOR pathway, which governs protein synthesis, growth control, and lifespan regulation in response to nutrient availability. Inhibiting the mTOR pathway extends lifespan in various organisms. However, the downstream effectors for the regulation of cell homeostasis that are critical to lifespan extension remain elusive. Here we show that fission yeast Maf1 is required for lifespan extension. Maf1's function in tRNA repression is inhibited by mTOR-dependent phosphorylation, whereas Maf1 is activated via dephosphorylation by protein phosphatase complexes, PP4 and PP2A. Mutational analysis reveals that Maf1 phosphorylation status influences lifespan, which is correlated with elevated tRNA and protein synthesis levels in maf1∆ cells. However, mTOR downregulation, which negates protein synthesis, fails to rescue the short lifespan of maf1∆ cells, suggesting that elevated protein synthesis is not a cause of lifespan shortening in maf1∆ cells. Interestingly, maf1∆ cells accumulate DNA damage represented by formation of Rad52 DNA damage foci and Rad52 recruitment at tRNA genes. Loss of the Rad52 DNA repair protein further exacerbates the shortened lifespan of maf1∆ cells. Strikingly, PP4 deletion alleviates DNA damage and rescues the short lifespan of maf1∆ cells even though tRNA synthesis is increased in this condition, suggesting that elevated DNA damage is the major cause of lifespan shortening in maf1∆ cells. We propose that Maf1-dependent inhibition of tRNA synthesis controls fission yeast lifespan by preventing genomic instability that arises at tRNA genes.

Keywords: DNA damage; DNA repair; Maf1; RNA polymerase III; aging; lifespan; tRNA; transcription.

Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
Loss of Maf1 shortens chronological lifespan. (a) WT and maf1Δ cells were cultured in YES medium containing 3% or 0.1% glucose overnight at 30°C. Levels of the pre‐tRNAser11‐met07 and act1 + genes were examined by RT–PCR from total RNA preparation. tRNA samples were run on acrylamide gels and stained by SYBR Green. (b) WT and maf1Δ cells were cultured overnight in YES medium with 0.1% or 5.0% glucose. Expressions of the pre‐tRNALeu05 and tRNALys01 were examined by RT–PCR. The histograms show the relative tRNA expression levels normalized to act1+ expression. Data are expressed as the mean of three independent experiments. Error bars represent the standard error of the mean (SEM). (c) WT and maf1Δ cells were first cultured at 30°C in YES liquid medium with different percentages of glucose (0.1%, 1%, and 5%) for the indicated days. Fivefold serial dilutions of the cells were then plated on YES agar medium containing 3% glucose, incubated for 3 days at 30°C, and photographed. The complete presentation of the results is shown in Figure S2. (d) Quantification of the lifespan assays shown in Figure S2 was performed by using NIH Image J. The average growth intensity of each strain on each day was obtained from colonies derived from six dilutions. To calculate the average deviation (error bar), two strains for each genotype were tested, and their growth was quantified
Figure 2
Figure 2
Maf1 is regulated by PP2A/PP4 phosphatases and the TORC1 kinase. (a, b) Cells of the indicated genotypes were engineered to express Maf1–5FLAG and grown at 30°C in YES liquid medium supplemented with 0.1%, 1%, or 5% glucose. Protein extracts were prepared, and Western blotting of Maf1‐5FLAG was performed. Tubulin was used as a loading control. Representative images of repeat experiments are shown. (c) WT, torc1, and maf1Δ cells were cultured in YES medium with different percentages of glucose (0.1%, 1%, and 5%) at 25°C for the indicated days. Fivefold serial dilutions of the cells were then plated on YES agar medium containing 3% glucose for lifespan assays. (d) Lifespan assay was performed with cells of the indicated genotypes. Cells were cultured in YES liquid medium supplemented with 0.1% glucose at 30°C for the indicated days before plating on YES agar medium as described above. Representative images of repeat experiments are shown
Figure 3
Figure 3
Maf1 phosphorylation dictates Schizosaccharomyces pombe lifespan and tRNA levels. (a) maf1Δ cells expressing Maf1–FLAG with the indicated mutations were cultured in YES medium with 3% glucose at 30°C. Equal amounts of protein were subjected to Western blotting and probed by the anti‐FLAG antibody. The mutations introduced in each Maf1 mutant are also indicated. (b) maf1Δ cells expressing Maf1–FLAG with the indicated mutations were cultured in YES liquid medium supplemented with 0.1% glucose at 30°C for the indicated days. For the lifespan assay, fivefold serial dilutions of cells were then plated on YES agar medium containing 3% glucose to evaluate viability. YES agar plates were incubated for 3 days at 30°C and photographed. (c) maf1Δ cells expressing Maf1–FLAG, Maf1‐7A–FLAG, and Maf1‐7E–FLAG were cultured in YES liquid medium supplemented with 0.1% glucose at 30°C. Expressions of the pretRNAser01‐met01, pre‐tRNAleu06, and pre‐tRNAarg05 were examined. tRNA expression was normalized to act1 + gene expression. Data are expressed as the mean of three independent experiments. Error bars represent standard error of the mean (SEM). (d, e) Cells of the indicated genotypes were cultured in YES liquid medium supplemented with 0.1% glucose at 30°C. tRNA expression analyses are performed as described in (b). (f) Lifespan assay of cells with the indicated genotypes was performed as described in (b). The complete presentation of the results is shown in Figure S5a
Figure 4
Figure 4
Maf1 is a major target of TORC1 in lifespan regulation. (a) Cells of the indicated genotypes were grown at 30°C in YES liquid medium supplemented with the indicated concentrations of glucose. Cells were then incubated with Trans‐35S for 15 min, and protein synthesis rate was expressed as the amount of radioactivity (counts per minute, CPM) incorporated in proteins. Error bars represent SEM obtained from three independent experiments. (b) WT, maf1Δ, torc1, and maf1Δ torc1 cells were cultured in YES liquid medium with different percentages of glucose at 25°C for the indicated days, and viability was assayed on YES agar medium containing 3% glucose at 25°C. (c) WT and maf1Δ cells were cultured in YES medium with different percentages of glucose in the presence or absence of rapamycin. Cultures were kept at 30°C for the indicated days, and viability was assayed as described in (b) at 30°C
Figure 5
Figure 5
Maf1 cells accumulate DNA damage causing lifespan shortening. (a, b) The indicated cells expressing Rad52‐YFP were grown to mid‐log phase in YES liquid medium with 0.1% glucose at 25°C. The cells were then subjected to fluorescence microscopy. At least 200 cells were counted for each strain. The percentages of nuclei with at least one Rad52‐YFP focus are shown. Error bars correspond to SEM obtained from three independent experiments. (c) Representative microscopic images of the indicated cells are shown. (d, e) Cells of the indicated genotypes were cultured in YES liquid medium with different percentages of glucose (0.1% and 1%) at 25°C (in d) or 30°C (in e) for the indicated days. Lifespan assay was performed by plating fivefold serial dilutions of cells on YES agar medium containing 3% glucose. The agar plates were photographed after 3–5 days of incubation at 25°C (in d) or 30°C (in e)
Figure 6
Figure 6
Maf1 prevents DNA damage at tRNA genes. (a, b) ChIP assays showing Rad52 enrichment at the indicated tRNA genes in wild‐type and maf1∆ cells. Rad52‐12Pk was chromatin‐immunoprecipitated from the indicated cells, and associated DNA was analyzed by competitive multiplex PCR to amplify DNA sequences from the indicated tRNA gene and a gene‐free region (GFR). GFR was used as an internal PCR amplification control as described in our previous study (Gadaleta et al., 2016). In (a), chromatin association of Rad52‐12Pk at the tRNA genes was presented as relative enrichment over the association at GFR. Blue bars indicate relative enrichments of Rad52 in the presence of 0.1% glucose, while orange bars show Rad52 enrichments in the presence of 5% glucose. Data are expressed as the mean of three independent experiments, and error bars correspond to SEM. In (b), Rad52‐12Pk association was presented as relative enrichment over the enrichment in wild‐type cells grown in the presence of 0.1% glucose

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