RAS-dependent ROS signaling in C. elegans.

Mitochondrial superoxide produced by low concentrations of the pro-oxidant paraquat increases C. elegans lifespan. We found that paraquat acts by intensifying RAS-dependent ROS signaling (RDRS) to alter the expression of >50% of the genome and control animal composition and physiology. We show that RDRS is regulated by negative feedback from SOD-1-dependent conversion of superoxide into cytoplasmic hydrogen peroxide, which in turn acts on a redox-sensitive cysteine (C118) of the RAS homologue LET-60ras. Preventing C118 oxidation by replacement with serine, or mimicking oxidation by replacement with aspartic acid, leads to opposite changes in the expression of thousands of genes. We show that paraquat acts through RDRS to exacerbate and accelerate gene expression changes that are normally observed at the end of post-embryonic development. The identities of the genes affected by paraquat suggest that RDRS stimulation extends lifespan by increasing turnover and repair while moderating damage from metabolic activity. Three biological replicates per condition were analysed; RNA was isolated individually from each replicate. C118S and C118D were sequenced in separate experiments and therefore contain their own WT controls for comparison (N2_Control_replicate 1 - replicate 3 are the WT controls used for C118S and N2_Control_replicate 4 – replicate 6 are the WT controls used for C118D). Bacteria grown on Nematode Growth Media (NGM) plates were transferred to experimental NGM plates for Paraquat (PQ) experiments (for both the PQ-treated and untreated controls). In contrast, the bacterial culture was seeded directly onto plates for developmental experiments. Therefore, the N2_gravid adults, N2_young adults and N2_L4 larvae samples should be compared to each other rather than to the N2_Control samples.