Description

Various non-coding RNAs (ncRNAs) change their levels during aging, and influence lifespan in many species, including Caenorhabditis elegans (Garg and Cohen 2014; He et al. 2018; Kim and Lee 2019, Kim et al. 2021). However, the role of Y RNAs, ncRNAs that regulate DNA replication and RNA quality control (Kowalski and Krude 2015), in aging and lifespan remains unknown. C. elegans genome encodes 19 Y RNA homologs, C. elegans Y RNA (CeY) encoded by yrn-1 and 18 stem-bulge RNAs (sbRNAs), each of which contains a conserved stem-loop structure (Fig. 1A) (Kowalski and Krude 2015). Y RNAs form a Ro60 ribonucleoprotein by binding Ro 60-kDa protein (Ro60), which is conserved from bacteria to human (Sim et al. 2020). ROP-1, the C. elegans Ro60, binds and stabilizes CeY (Van Horn et al. 1995; Labbé et al. 1999). rop-1 mRNA and ROP-1 protein levels increase during aging in C. elegans (Liang et al. 2014; Mansfeld et al. 2015; Angeles-Albores et al. 2017), suggesting the potential aging-regulatory functions of ROP-1 and its associated RNA, CeY. Here, we sought to determine the role of the Y RNA homologs in aging and lifespan.

We first tested whether the levels of C. elegans Y RNA homologs changed during aging by performing real-time quantitative reverse transcription PCR (qRT-PCR) analysis. We found that the levels of the CeY and sbRNAs were generally increased during aging (Fig. 1B). In addition, we showed that the mRNA level of rop-1 was increased during aging (Fig. 1B), consistent with previous reports (Mansfeld et al. 2015; Angeles-Albores et al. 2017).

Increased stress resistance is associated with longevity in multiple species (Epel and Lithgow 2014, Park et al. 2017), and stress-responsive factors play crucial roles in aging (Son et al. 2019). We therefore measured the effect of oxidative or thermal stresses on the levels of C. elegans Y RNA homologs. We found that the level of CeY was decreased under both oxidative (Fig. 1C) and thermal stress conditions (Fig. 1D). We also showed that oxidative stress significantly increased the levels of four sbRNAs, Ce4, Ce5, CeN75, and CeN73-1 (Fig. 1C), whereas thermal stress did not (Fig. 1D; see Figure 1D legend for discussion). In contrast to the decrease in the level of CeY under these tested stress conditions, the mRNA level of rop-1 was increased by oxidative stress (Fig. 1C) but not by thermal stress (Fig. 1D). Thus, it seems likely that these two external stresses affect the expression of various C. elegans Y RNA homologs and rop-1 differently.

We then focused our functional analysis on CeY by using CRISPR/Cas9 gene targeting, because the potential functional redundancy of 18 sbRNAs may hinder characterizing the roles of individual sbRNAs in aging. We generated two mutant alleles, yh84 and yh85, in yrn-1 gene that encodes CeY (Fig. 1E). We showed that yh84 or yh85 mutation substantially reduced the level of CeY (Fig. 1F). In addition to these yrn-1 mutants, we characterized two different rop-1 mutant alleles, rop-1(pk93) and rop-1(ok2690), both of which substantially reduced the level of CeY and rop-1 mRNA (Fig. 1F, G) (Labbé et al. 1999). We then measured the lifespan of these mutants, and found that yrn-1(yh84) or yrn-1(yh85) mutation did not affect the lifespan of wild-type animals at 20°C (Fig. 1H, I, M, N). Reducing insulin/IGF-1 signaling via inhibition of daf-2/insulin/IGF-1 receptor doubles lifespan (Kenyon et al. 1993), and is one of the most studied longevity-promoting interventions in C. elegans (Altintas et al. 2016). A major function of insulin/IGF-1 signaling in C. elegans is to regulate the proper formation of dauer, an alternative hibernation-like developmental stage (Hu 2007). rop-1(pk93) mutations decrease dauer formation in a wild-type background, and enhance or suppress the increased dauer formation by daf-2(-) mutants in an allele-specific manner (Labbé et al. 2000). These findings raise the possibility that rop-1 and/or CeY affects physiological processes regulated by insulin/IGF-1 signaling. However, yrn-1(yh84) or yrn-1(yh85) mutation had little effect on the lifespan of daf-2 RNAi-treated worms (Fig. 1H, I, M, N). Therefore, CeY appears to be dispensable for maintaining normal lifespan and promoting longevity by reduced insulin/IGF-1 signaling. We also found that rop-1(pk93) mutations decreased the lifespan of both control RNAi- and daf-2 RNAi-treated worms (Fig. 1J, M, N), whereas rop-1(ok2690) mutation did not (Fig. 1K, M, N). A previous study reported that rop-1(pk93) mutations do not affect lifespan (Labbé et al. 1999). We found that rop-1(pk93) mutations substantially increased vulval rupturing during adulthood (Fig. 1L), which reduces lifespan (Leiser et al. 2016). Thus, mutations in rop-1 do not appear to directly affect aging per se but may cause early deaths during adulthood by increasing vulval rupture phenotypes in our experimental conditions. Together, we did not find apparent lifespan phenotypes in yrn-1 or rop-1 mutants, both of which displayed substantially reduced CeY levels.

Our study points to the possibility that CeY may not directly participate in the regulation of lifespan. CeY alone may not be functionally equivalent to the vertebrate Y RNA, as CeY does not affect DNA replication, likely because of the lack of conserved motif in the upper stem (Gardiner et al. 2009; Boria et al. 2010; Kowalski et al. 2015). In contrast, sbRNAs contain a conserved UG-CA motif (Fig. 1A) and participate in DNA replication (Kowalski et al. 2015). Thus, it will be important to test whether the inhibition of sbRNAs affects lifespan. In particular, investigating the roles of four oxidative stress-induced sbRNAs, Ce4, Ce5, CeN75, and CeN73-1, which were also age-dependently upregulated, in aging and lifespan will be crucial for future research.