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Certain RNA interference (RNAi) responses are passed from parent to progeny and can persist across multiple generations. It has generally been assumed that the fading of these 'memories' over a few generations is a passive process attributable to dilution of an inherited factor. Now, a paper published in Cell reports that heritable silencing in Caenorhabditis elegans is under the control of an active tunable feedback mechanism — a 'transgenerational timer' that dictates whether ancestral RNAi responses are memorized and for how long.

The authors hypothesized the existence of a feedback mechanism based on their observation that heritable endogenous small interfering RNAs (endo-siRNAs) align antisense to, and hence regulate, endo-siRNA biogenesis genes. They reasoned that exogenous RNAi could potentially activate such a feedback mechanism, switching off the RNAi inheritance machinery in progeny and thereby controlling the duration of heritable silencing.

To explore this possibility, the team used transgenically encoded GFP as a target for exogenous double stranded RNA (dsRNA)-induced RNAi. Exposing worms to anti-gfp dsRNA resulted in the silencing of GFP in treated worms and progeny, which 'petered out' after four generations. Surprisingly, the subsequent exposure of progeny to a 'second dsRNA trigger' (targeting a different gene unrelated to gfp) extended the transgenerational persistence of the inherited gfp silencing. Furthermore, exposure to the second dsRNA trigger in consecutive generations prolonged and enhanced gfp silencing for additional generations, supporting the idea that the decay of heritable RNAi is actively regulated and not merely due to dilution.

Consistent with the phenotypic extension of GFP silencing, RNA sequencing (RNA-seq) experiments revealed that exposure to a second trigger significantly amplified the level of secondary anti-gfp siRNAs in second- and third-generation progeny. Thus, a specific dsRNA trigger can lead to the amplification of other unrelated small RNAs, suggesting an overall effect on the functionality of the RNAi system.

The authors sought to determine whether exogenous RNAi extends transgenerational silencing by affecting the levels of heritable endo-siRNAs that regulate endo-siRNA biogenesis genes. Analysis of RNA-seq data revealed significant downregulation of several endo-siRNA pathways following dsRNA-induced exogenous RNAi, consistent with previous evidence of competition between exogenous and endogenous siRNA pathways.

Targets of heritable endo-siRNAs that were differentially expressed following dsRNA exposure were enriched for genes involved in epigenetic regulation and included genes encoding factors known to be required for RNAi inheritance. Mutation of these genes altered the normal duration of heritable RNAi response — a phenotype that the authors term MOTEK (modified transgenerational epigenetic kinetics).

Based on these experiments, the authors propose that the RNAi machinery switches between different states that enable or restrict exogenous siRNA (exo-siRNA) inheritance. Exogenous RNAi turns the system on by amplifying exo-siRNAs at the expense of endo-siRNAs, and the system is turned off via a feedback pathway in which the endo-siRNA- mediated regulation of genes required for inheritance of endo-siRNAs is altered. Environmentally induced RNAi responses acting as 'second triggers' reset this transgenerational timer, extending the duration of heritable silencing. The authors suggest that this is an evolved, adaptive mechanism that controls the inheritance of ancestral responses that could be harmful in progeny that are subject to environmental conditions that differ from those of their parents.