Deletion of microRNA-80 Activates Dietary Restriction to Extend C. elegans Healthspan and Lifespan
Figure 6
CBP-1 is critical for mir-80(Δ) healthspan benefits, and is a candidate direct binding target of miR-80.
Fig. 6A. cbp-1(RNAi) in the mir-80(Δ) background reverses the DR Exmax shift. We grew age-synchronized animals under standard RNAi feeding conditions (20°C, HT115) and measured age pigments at Day 4 (50 animals per RNAi clone). We recorded Exmax as the highest peak detected by the Datamax software package suite (Horiba Scientific). Graphs represent cumulative data from 3 independent trials. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (** p<0.001, * p≤0.055 compared to mir-80(Δ) empty vector). cbp-1(RNAi) Exmax is comparable to that of ad lib wild type (p = 0.729). Note that cbp-1(RNAi) treatment of WT does not change Exmax (data not shown), so this effect is specific to the DR signature of mir-80(Δ). Fig. 6B. cbp-1(RNAi) in the mir-80(Δ) background partially reverses low age pigment levels. We grew age-synchronized animals under standard conditions (20°C, HT115) and measured total age pigment fluorescence at day 4 (50 animals per RNAi clone), normalized to total tryptophan fluorescence as in ref. [19]. Graphs represent cumulative data from 3 independent trials. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (** p<0.05, * p<0.1 compared to mir-80(Δ)+empty vector RNAi). Note that cbp-1(RNAi) treatment of WT induces modest reduction of age pigment levels (p = 0.01, data not shown). Fig. 6C. mir-80(Δ) longevity is dependent on cbp-1. We placed age-synchronized L1 larvae on empty vector control (pL4440) plates under standard conditions (20°C) until Day 4 (day 1 of adult life) at which time animals were moved to either empty vector control (L4440) or cbp-1(RNAi) plates. At day 9, we placed 10 healthy animals per plate (≥40 per strain per trial), and we scored viability as movement away from pick touch on the indicated days. The graphs represent data combined from 3 independent trials. Statistics are calculated using the Log-rank Test. cbp-1(RNAi) decreases the lifespan of mir-80(Δ) (p<0.0001 compared to vector control. Because RNAi knockdown is inefficient the nervous system (see [59]), the profound effects of cbp-1(RNAi) suggest that critical cbp-1/mir-80 regulation occurs outside of the C. elegans nervous system. Fig. 6D. The cbp-1 transcript includes two predicted binding sites for miR-80. Exon structure of cbp-1 is indicated by thick blue bars, introns in thin black lines (see WormBase for details). The rna22 algorithm [10], which searches for target sites outside the 3′UTR, predicts that miR-80 binds cbp-1 within the 5′ UTR and within exon 8. The potential alignments of miR-80 (red) to C. elegans cbp-1 (blue) sequences are indicated. Note that the seed match to the exon 8 region is a perfect 10 bp match for C. elegans, and that the target sequence is conserved in mouse and human CBP1 (see Fig. S7). Fig. 6E. Endogenous CBP-1 protein levels are increased in 7 day old mir-80(Δ) mutants. We grew age-synchronized animals under standard conditions (20°C, OP50-1) and extracted total protein at Day 7 (100 animals per strain) for Western blot analysis (top). Graphs represent CBP-1 levels for each strain normalized to own TUB-1 levels. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (** p<0.005). The graphs represent data combined from 3 independent trials. We noted that during young adulthood, native levels of CBP-1 seemed comparable to WT in mir-80(Δ), suggesting that additional regulatory controls are exerted on CBP-1 expression levels in development or early adulthood.