The 18S rRNA methyltransferase DIMT-1 regulates lifespan in the germline later in life

Abstract

Specialized ribosomes help determine which proteins are synthesized, however, the influence of age on ribosome heterogeneity and whether dysregulation of this process drives organismal aging is unknown. Here we examined the role of ribosomal RNA (rRNA) methylation in maintaining appropriate translation as organisms age. In a directed RNAi screen, we identified 18S rRNA N6'-dimethyl adenosine (m^6,2A) methyltransferase, dimt-1, as a regulator of C. elegans lifespan and stress resistance. We demonstrate that DIMT-1 functions in the germline after mid-life to regulate lifespan. Depletion of dimt-1 leads to selective translation of transcripts important for stress resistance and lifespan regulation in the C. elegans germline including the cytochrome P450 daf-9, which synthesizes a steroid that signals from the germline to the soma. dimt-1 induced lifespan extension is dependent on the daf-9 signaling pathway. Our findings highlight ribosome heterogeneity, and specific rRNA modifications, in maintaining appropriate translation later in life to promote healthy aging.

Fig. 1. Ribosomal RNA methyltransferases regulate lifespan, heat, and UV stress response.

a–c Directed RNAi screen of putative rRNA methyltransferases reveals changes in a lifespan, b UV stress survival, and c heat stress survival relative to empty vector control worms. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Each dot represents an independent experiment with 30 worms per plate in three plates. One-way ANOVA with Dunnett’s multiple comparisons was used to calculate statistics within a single experiment for stress assays, and log-rank (Mantel–Cox) test were performed for longevity assays. Fisher’s combined method was used to calculate p values across multiple independent experiments. All columns represent the mean ± SEM (a) or SD (b, c). Some RNAi clones were not replicated (without error bars) due to no effect being observed.

Fig. 2. Ribosomal RNA modifications in 26S and 18S are dynamically regulated throughout life and change in response to UV and heat stress.

a 26S and 18S rRNA extracted from C. elegans at different ages, from 4 days to 19 days, reveal changes in rRNA methylation as assessed by UHPLC-ms/ms. This heat map represents the relative changes of methylation in 4 biological replicates. Changes in individual modifications can be seen in Fig. S2C. b, c 26S and 18S rRNA extracted from C. elegans after UV stress (b) or 37 °C heat stress (c) relative to worms not exposed to UV (ctl) or grown at 20 °C reveal some changes in rRNA methylation as assessed by UHPLC-ms/ms. These heatmaps represent the relative changes of methylation in three biological replicates. Changes in individual modifications can be seen in Supplementary Fig. 2e, f.

Fig. 3. Longevity induced by dimt-1 deficiency requires the FoxO and TOR signaling pathways and requires an intact germline.

a Mutation of glutamic acid 79 to an alanine (E79A) in dimt-1 caused a complete elimination of 18S rRNA m^6,2A as assessed by UHPLC-ms/ms. Statistics represent an unpaired two-tailed t-test with Welch’s correction. b Mutation of E79A in dimt-1 caused lifespan extension relative to WT worms. c dimt-1 knockdown extends the lifespan of both WT and eat-2(ad1116) mutant worm lifespan to a similar extent (p = 0.7021 by two-way ANOVA). d dimt-1 knockdown extends the lifespan of both WT and daf-2(e1370) mutant worm lifespan to a similar extent (p = 0.0806 by two-way ANOVA). e dimt-1 knockdown extends the lifespan of both WT and hsf-1(sy441) mutant worm lifespan to a similar extent (p = 0.4245 by two-way ANOVA). f dimt-1 knockdown extends the lifespan of WT but not daf-16(mu86) mutant worm lifespan (p < 0.0001 by two-way ANOVA). g dimt-1 knockdown extends the lifespan of WT but does not further extend the long lifespan of raga-1(ok386) mutant worms (p = 0.0327 by two-way ANOVA). h dimt-1 knockdown extends the lifespan of WT but does not further extend the long lifespan of germline deficient glp-1(e2141ts) mutant worms that were shifted to the restrictive temperature at the L1 stage (p < 0.0001 by two-way ANOVA). i dimt-1 knockdown extends the lifespan of WT but not sterile pgl-1(bn101ts) mutant worms whose mothers were shifted to the restrictive temperature (25.5 °C) at the L4 stage (p < 0.0001 by two-way ANOVA). Statistics and replicate experiments are presented in Supplementary Table 1. ns not-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as calculated by log-rank (Mantel–Cox) statistical test.

Fig. 4. DIMT-1 functions in the germline to regulate lifespan and affects the translation of specific mRNA transcripts.

a Auxin-inducible degron (AID) ubiquitous degradation of DIMT-1 protein leads to significant decrease in m^6,2A levels in the 18S rRNA subunit as assessed by UHPLC-ms/ms. Statistics represent an unpaired two-tailed t-test p = 0.0286. b Ubiquitous and germline-specific AID-induced DIMT-1 protein degradation causes a lifespan extension, while DIMT-1 depletion in the muscle, intestine, or neurons has no effect on lifespan extension. c, d Tissue-specific knockdown of dimt-1 in the germline increases lifespan to a similar extent as in ubiquitous knockdown, while knockdown of dimt-1 in the muscle, intestine, or neurons has no significant effect on longevity. e dimt-1 knockdown does not extend the lifespan of worms treated with 5-fluorodeoxyuridine (FUdR), a drug that inhibits the proliferation of germline stem cells and the production of intact eggs. Statistics and replicate longevity experiments are presented in Supplementary Table 2. ns not-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001 as calculated by log-rank (Mantel–Cox) statistical test.

Fig. 5. DIMT-1 affects the translation of specific mRNA transcripts.

a Heatmaps of the 2082 differentially ribosome-bound transcripts in the ribosome after dimt-1 knockdown from day 7 worms. Ribosome binding was normalized to total RNA expression to give translation efficiency. Dimt-1 was knocked down from the L4 stage until day 7. Each column represents an independent biological replicate from ribosome sequencing after TRAP. b Pathway analysis of differentially bound transcripts after dimt-1 knockdown revealed altered ribosome binding to transcripts involved in longevity regulation, degradation, fatty acid metabolism, the ribosome, and oxidative phosphorylation. RNAseq and translation efficiency significantly regulated genes and gene ontology categories are presented in Supplementary Data 1, 2. c Sequence motifs enriched in the 5’ UTR of more bound mRNA transcripts after dimt-1 knockdown. d dimt-1 knockdown extends the lifespan of WT but not daf-9 mutant worms (p = 0.004 by two-way ANOVA). e dimt-1 knockdown extends the lifespan of WT but not daf-12 mutant worms (p = 0.0005 by two-way ANOVA).

Fig. 6. DIMT-1 regulates lifespan after mid-life.

a Experimental design for the AID-tagged DIMT-1 temporal knock out experiments (y.a. young adults). Red lines indicate when strains were placed on 150 uM auxin and when DIMT-1 is knocked down. b AID-induced depletion of DIMT-1 in the germline extends lifespan when depleted in the previous generation and for the entirety of the assayed generation, starting at birth, or from young adulthood for the remainder of the lifespan but does not extend lifespan when depleted only from birth until young adulthood. c AID-induced depletion of DIMT-1 ubiquitously extends lifespan when depleted in the previous generation and for the entirety of the assayed generation, starting at birth, or from young adulthood for the remainder of the lifespan and causes a less dramatic extension in lifespan when depleted only from birth until young adulthood. d AID-induced depletion of DIMT-1 in the germline extends lifespan to a similar extent when depleted from young adulthood, after reproduction, or starting in mid-life. Statistics and replicate longevity experiments are presented in Supplementary Table 3. ns not-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001 as calculated by log-rank (Mantel–Cox) statistical test.