Three Rules Explain Transgenerational Small RNA Inheritance in C. elegans

Life experiences trigger transgenerational small RNA-based responses in C. elegans nematodes1. Dedicated machinery ensures that heritable effects would re-set, typically after a few generations2,3. Here we show that isogenic individuals differ dramatically in the persistence of transgenerational responses. By examining lineages composed of >20,000 worms we reveal 3 inheritance rules: (1) Once a response is initiated, each isogenic mother stochastically assumes an “inheritance state”, establishing a commitment that determines the fate of the inheritance. (2) The response that each mother transfers is uniform in each generation of her descendants. (3) The likelihood that an RNAi response would transmit to the progeny increases the more generations the response lasts, according to a “hot hand” principle. Mechanistically, the different parental “inheritance states” correspond to global changes in the expression levels of endogenous small RNAs, immune response genes, and targets of the conserved transcription factor HSF-1. We show that these rules predict the descendants’ developmental rate and resistance to stress. Four mCherry-expressing worms were allowed to lay eggs in separate plates (one worm per plate) for 8 hours. The eggs were allowed to hatch and grew until they reached adulthood. Each group of progenies (isogenic sisters) was then washed with M9 buffer into a 1.5ml Eppendorf tube, followed by 3-4 M9 washes, in order to remove any residual bacteria. Each group was then examined in COPASTM Biosort (Union Biometrica) and sorted to get the top and bottom 25% of mCherry expressing worms in each group. TRIzol® (Life Technologies) was then added to each sorted tube, and the tubes were immediately transferred to -80°C until RNA extraction procedure.