RNA-processing factors with altered expression during physiological aging contribute to the longevity of daf-2 mutants. (A) Normalized enrichment of expression changes in genes that encode components annotated as RNA processing (GO:0006396) in aged animals. Adjusted P values are shown at the top of the data points, calculated by gene set enrichment analysis test and adjusted using false discovery rates; (***) adjusted P < 0.001. (B) Significance of the likelihood-ratio test for 11 genes that encode RNA-processing components and were down-regulated during aging specifically in wild-type (WT) but not in daf-2(e1370) [daf-2(-)] animals. (C–F) Expression changes of prp-8 (C), plrg-1 (D), pfs-2 (E), and F30A10.9 (F) during aging in WT and daf-2(-) animals. Adjusted P values are shown, calculated by using DESeq2's Wald test relative to day 1 of adulthood and adjusted by using the procedure of Benjamini and Hochberg; (*) adjusted P < 0.05, (**) adjusted P < 0.01, (***) adjusted P < 0.001. (G) Comparison of lifespan changes conferred by RNAi targeting each of the eleven genes in B and daf-16 (a positive control, purple symbols) in WT and that in daf-2(-) animals. Red symbols represent RNAi clones that shortened the lifespan of daf-2(-) animals by at least 10% more than that of WT animals, whereas a blue symbol represents a condition that shortened the lifespan of WT animals by at least 10% more than that of daf-2(-) animals. Whole-life RNAi treatment was used except those noted with triangles, which were performed using adult-only RNAi treatment. On excluding prp-8 RNAi data, an outlier that deviated from normal Q-Q line and Cook's distance (> 4/n = 0.14; n = 28), our results indicate that the effects of RNAi clones on lifespan were proportionally larger in daf-2(-) animals than in WT animals (y = 1.42x, simple linear regression without an intercept, adjusted R2 = 0.89). These data are consistent with the age-dependent changes in the expression levels of the candidate genes whose down-regulation was greater in WT than in daf-2(-) animals. (H–K) Lifespan (n ≥ 90 for each condition) of WT and daf-2(-) animals treated with control RNAi and RNAi targeting each of prp-8 (H), plrg-1 (I), pfs-2 (J), and F30A10.9 (K). See Supplemental Source files for additional repeats and statistical analysis of the lifespan data shown in this figure.
