Description

Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase, regulates biological processes in response to changes in nutrients or metabolites (Reviewed in Liu and Sabatini, 2020). Inhibition of mTOR signaling delays aging and increases lifespan in multiple organisms by downregulating substrates, including S6 kinase (S6K) that phosphorylates ribosomal subunit 6 (Reviewed in Kennedy and Lamming, 2016). Genetic inhibition of RSKS-1, the Caenorhabditis elegans S6 kinase, extends lifespan via decreasing protein synthesis and upregulating longevity-promoting factors (Reviewed in Johnson et al., 2013; Lee et al. 2015). However, the longevity phenotypes of previously characterized loss-of-function rsks-1 mutants are variable and greatly affected by environmental factors, such as temperatures (Seo et al., 2013; Miller et al., 2017). Here, we aimed at identifying rsks-1 mutants whose lifespan phenotypes are robust and reproducible at 20°C, the standard temperature for C. elegans lifespan assays.

We measured the lifespan of three available rsks-1 missense mutants generated by the Million Mutant Project (Thompson et al., 2013) after outcrossing four times to wild-type (N2) strains, without treatment with 2′-deoxy-5-fluorouridine. The three missense mutant alleles are rsks-1(gk185793), rsks-1(gk587386), and rsks-1(gk902595), which respectively cause E255 to K, P360 to S, and D370 to N changes (Fig. 1A–D). We also measured the lifespan of previously characterized rsks-1(tm1714) deletion mutants (Fig. 1A) (Chen et al., 2013; Seo et al., 2013). We found that rsks-1(gk185793), which encodes RSKS-1 E255K, caused a consistent and strong longevity phenotype in all three independent trials (Fig. 1E and I). In contrast, rsks-1(gk587386) and rsks-1(tm1714) mutations extended lifespan in one out of two trials (Fig. 1F, G, and I). We found that rsks-1(gk902595) lengthened lifespan slightly (p<0.01) or robustly (p<0.0001), or did not (p=0.2997) in three trials (Fig. 1H and I). These variations in the lifespan of the three latter rsks-1 mutants (Fig. 1F–I) may have been caused by small changes in environmental factors such as temperatures that affect the lifespan of rsks-1 mutants (Seo et al., 2013; Miller et al., 2017). Together, our data indicate that rsks-1(gk185793) mutant allele that alters the acidic E255 residue to a basic K increases the lifespan of C. elegans reproducibly and robustly.

We speculate that the effects of rsks-1(gk185793) on lifespan were more robust than other missense alleles tested, because of following two reasons. First, E255 resides in the catalytic kinase domain of RSKS-1/S6K, but the others are located in the AGC-kinase C-terminal domain (Fig. 1A–D). Second, E to K, a change from an acidic to a basic amino acid, is likely to cause a bigger impact on the protein function than P to S and D to N changes. Future studies with additional missense mutations will clarify this issue.

Our study suggests that rsks-1(gk185793) mutant C. elegans is an excellent tool for aging research as a reliable genetic model for longevity conferred by reduced mTOR signaling. Additional future studies will enhance the value of rsks-1(gk185793) mutants for research on aging and mTOR signaling. First, further outcross of rsks-1(gk185793) mutants to wild-type worms to remove remaining background mutations will corroborate the causality of change in RSKS-1 for longevity. Alternatively or additionally, generation of the identical missense mutant allele by using CRISPR genome editing will be helpful. Second, confirming whether rsks-1(gk185793) is a reduction-of-function allele will be crucial for establishing the rsks-1 mutant allele as a bona fide genetic model for reduced mTOR signaling. To this end, the assessment of phosphorylated ribosomal S6 and global protein synthesis levels will be required. Third, performing lifespan assays at various temperatures (e.g. 15°C and 25°C) and on different diets (e.g. E. coli HT115 and HB101 strains) may extend the utility of rsks-1(gk185793) mutants as a general longevity model. Lastly, E255 in C. elegans RSKS-1 is invariably conserved in mammalian S6K (Fig. 1D); therefore, it will be interesting to determine whether the orthologous E to K change in S6K extends lifespan in mice or is associated with human longevity.