Longer lifespan in the Rpd3 and Loco signaling results from the reduced catabolism in young age with noncoding RNA

Abstract

Downregulation of Rpd3 (histone deacetylase) or Loco (regulator of G-protein signaling protein) extends Drosophila lifespan with higher stress resistance. We found rpd3-downregulated long-lived flies genetically interact with loco-upregulated short-lived flies in stress resistance and lifespan. Gene expression profiles between those flies revealed that they regulate common target genes in metabolic enzymes and signaling pathways, showing an opposite expression pattern in their contrasting lifespans. Functional analyses of more significantly changed genes indicated that the activities of catabolic enzymes and uptake/storage proteins are reduced in long-lived flies with Rpd3 downregulation. This reduced catabolism exhibited from a young age is considered to be necessary for the resultant longer lifespan of the Rpd3- and Loco-downregulated old flies, which mimics the dietary restriction (DR) effect that extends lifespan in the several species. Inversely, those catabolic activities that break down carbohydrates, lipids, and peptides were high in the short lifespan of Loco-upregulated flies. Long noncoding gene, dntRL (CR45923), was also found as a putative target modulated by Rpd3 and Loco for the longevity. Interestingly, this dntRL could affect stress resistance and lifespan, suggesting that the dntRL lncRNA may be involved in the metabolic mechanism of Rpd3 and Loco signaling.

Keywords: Rpd3 and Loco signaling; lifespan; noncoding RNA; reduced catabolism; stress resistance.

Figure 1

Rpd3 and Loco regulate stress resistance and lifespan overlappingly. (A) The survival curve for oxidative stress using 2-day-old male flies. Control: UAS-loco/+; Rpd3-Down: rpd3^- UAS-loco/+; Loco-Up: UAS-loco/actG4; Rpd3-Down+Loco-Up: rpd3^- UAS-loco/actG4; p-value: log-rank test between the control and Rpd3-Down or Loco-Up. (B) Changes of oxidative stress resistance. The median survival times of flies under oxidative stress were calculated from several survival curves (A) and then the percentage change from control flies (0%) are represented as average ± standard error of mean (SEM) following normalization with the median of control flies (21.4 hours). P-value (*): Student’s t-test. (C) The lifespan of adult male flies. (D) Percent changes of mean lifespan are indicated as average ± SEM normalized by the control’s mean lifespan (UAS-loco/+: 39.4 days), which were calculated from several lifespan curves (C) of 2 ~ 4 independent experiments. The mean lifespan of another control +/actG4 flies was 40.1 days as single transgenic. (E) Venn diagram of differential genes with RNA-seq analyses between the control and Rpd3-Down or Loco-Up. Gene A and B indicate the groups changed with Rpd3-Down and Loco-Up, respectively. The C represents commonly changed genes in both Rpd3-Down and Loco-Up.

Figure 2

Longer lifespan induced by the change of Rpd3 and Loco results from the reduced catabolism. (A) The gene expression analyses (Rpd3-Down or Loco-Up/control). From RNA-seq experiments using 2-day-old male flies, the genes changed more than 1.3-fold were selected with p-value <0.05 (Fisher’s exact test). The underlined genes represent that their expressions decrease (green) and increase (purple) in a different group, respectively. (B) With the genes oppositely changed in the Rpd3-Down and Loco-Up (*: total 39 in A), the gene ontology was analyzed using a DAVID web tool (http://david.abcc.ncifcrf.gov/home.jsp). %: involved genes/total 39 genes; p-value: a modified Fisher Exact. (C) Expressional changes of selected genes in RNA-seq and real-time PCR analyses. The fold changes were averaged from 4 ~ 13 independent experiments using four different RNA batches. Bold change: p-value < 0.05 ~ 0.0001. (D) Functional analyses (http://flybase.org) of genes which were selected with more than 1.5-fold changes and p-value <0.05 in both Rpd3-Down (longer lifespan) and Loco-Up (shorter lifespan) groups. *: genes tested as a representative of each functional subgroup with RT-PCR analysis in (C).

Figure 3

Noncoding RNAs related to the longevity of Rpd3 and Loco. (A) Expression analyses of the noncoding genes from RNA-seq experiments of Rpd3-Down or Loco-Up over the control. Among the genes with fold change > |1.3| and p-value < 0.05, the underline indicates that the expression decreases (green) and increases (purple) in each different group. (B) Expressional fold changes of selected genes with real-time PCR analyses (p-value < 0.02 ~ 0.0001). Loco-Dn: comparison between 2-day-old male flies of loco^-/+ and wild-type (+/+); Old-Age: expression in the old flies (7 weeks) over young (1 week) flies between wild-type males; ns: non-specific change; *: expression changes of coding genes tested in Figure 2D. (C) The survival curve for starvation stress using 2-day-old male flies. P-value: log-rank test between the control (+/actG4) and CR45923/actG4 flies. (D-F) Stress resistance changes against starvation (D), heat (E), and oxidation (F). The percentages of median survival times changed from the +/actG4 flies (0%) are represented as average ± SEM followed by calculation of the median from several stress survival curves. P-value (*): Student’s t-test; I-rpd3: inverted sequence of rpd3. (G) The lifespan of adult male CR45923/actG4 flies with the controls (+/actG4 and CR45923/+). (H) Percent changes of mean lifespan are indicated as average ± SEM normalized by the +/actG4’s mean lifespan (39.3 ~ 42.8 days), which were calculated from several lifespan curves (G) of 3 ~ 5 independent experiments and were also tested with another driver tublin-Gal4. Parentheses: changed percentage out of data range in the graph.