Figure 1 : Age-dependent changes in C. elegans Y RNA homologs, and the effects of
yrn-1 and
rop-1 mutations on lifespan. (A) A schematic showing the conserved structure of C. elegans Y RNA homologs, CeY RNA (CeY) and stem-bulge RNAs (sbRNAs). Each of CeY and sbRNAs contains a conserved cytidine bulge (Van Horn et al. 1995; Deng et al. 2006; Boria et al. 2010), a characteristic of Y RNAs. Each sbRNA also contains a highly conserved UG-CA motif. (B) A heat map showing fold changes in the levels of the CeY, sbRNAs, and
rop-1 mRNA in wild-type C. elegans at days 4, 7, and 11 of adulthoods compared to those in day 1 animals, which were analyzed using qRT-PCR [n=3 except for CeY (n=6),
rop-1 (n=5), CeN71 (n=5), and CeN72 (n=5)]. (C and D) qRT-PCR analysis for changes in the levels of CeY (tan), sbRNAs (gray), and
rop-1 mRNA (green) under oxidative stress [7.5 mM tert-butyl hydroperoxide (t-BOOH)] [(C), n=4 except for CeN74-2 (n=3), CeN75 (n=3), Ce4 (n=3), and Ce5 (n=3)] and thermal stress (33°C) conditions [(D), n=4]. Red dotted lines indicate relative RNA levels in control conditions, which are normalized to one. Inset scatter plots show relative RNA levels of CeY (dotted box). [We noticed that a part of our data in panel D are not consistent with a previous report (Deng et al. 2006). By using northern blot analysis, Deng et al. reported that the levels of several sbRNAs, including CeN71, CeN72, CeN73-2, CeN74-1, CeN76, and CeN77, were increased with heat shock (30°C) for 3 hours. In contrast, we treated worms with 33°C for 30 minutes, which may have caused the difference.] n: the number of biological repeats. P values were calculated by using two-tailed Student's t test (*P < 0.05, **P < 0.01). (E) A schematic showing the regions altered by
yrn-1(
yh84) and
yrn-1(
yh85) mutant alleles generated by CRISPR/Cas9-mediated genome editing.
yrn-1(
yh84) caused 35 base pair (bp) indel mutation (2 bp deletion and 37 bp insertion) at 4 bp upstream of
yrn-1.
yrn-1(
yh85) caused one bp deletion at the 5' end of
yrn-1, 51 bp deletion spanning 45 bp at the 3' end and 6 bp immediately downstream of
yrn-1, and 2 bp insertion immediately downstream of the 45 bp deletion site. See Methods for sequence information of
yrn-1(
yh84) and
yrn-1(
yh85) mutations. (F and G) qRT-PCR analysis for the effects of
yrn-1(
yh84) (n=4),
yrn-1(
yh85) (n=2),
rop-1(
pk93) (n=4), and
rop-1(
ok2690) (n=3) on the levels of CeY (F) and
rop-1 mRNA (G). Levels of CeY and
rop-1 mRNA in wild-type are normalized to one. P values were calculated by using two-tailed Student's t test (*P < 0.05, ***P < 0.001). (H-K) Pooled lifespan curves of wild-type (WT) and
yrn-1(
yh84) (H),
yrn-1(
yh85) (I),
rop-1(
pk93) (J), and
rop-1(
ok2690) (K) mutant animals treated with control RNAi or
daf-2 RNAi. (L) Percent animals that displayed vulval rupture phenotypes and that were subsequently censored during lifespan assays [panels H to K, n (trial number) =2]. P values were calculated by using two-tailed Student's t test (*P < 0.05, **P < 0.01). (M and N) Statistical analysis of individual replicates of lifespan assays (panels H to K). All percent lifespan changes and P values were calculated against WT animals within each control or
daf-2 RNAi-treated groups otherwise noted with superscripts. WT: percent lifespan change or P value against WT in a control RNAi condition within replicate. P values were calculated using Mantel-Cox log-rank test. s.e.m. indicates the standard error of mean.