An animal that can transfer gene-regulatory information from somatic cells to germ cells may be able to communicate changes in the soma from one generation to the next. In the worm Caenorhabditis elegans, expression of double-stranded RNA (dsRNA) in neurons can result in the export of dsRNA-derived mobile RNAs to other distant cells. Here, we show that neuronal mobile RNAs can cause transgenerational silencing of a gene of matching sequence in germ cells. Consistent with neuronal mobile RNAs being forms of dsRNA, silencing of target genes that are expressed either in somatic cells or in the germline requires the dsRNA-selective importer SID-1. In contrast to silencing in somatic cells, which requires dsRNA expression in each generation, silencing in the germline is heritable after a single generation of exposure to neuronal mobile RNAs. Although initiation of inherited silencing within the germline requires SID-1, a primary Argonaute RDE-1, a secondary Argonaute HRDE-1, and an RNase D homolog MUT-7, maintenance of inherited silencing is independent of SID-1 and RDE-1, but requires HRDE-1 and MUT-7. Inherited silencing can persist for >25 generations in the absence of the ancestral source of neuronal dsRNA. Therefore, our results suggest that sequence-specific regulatory information in the form of dsRNA can be transferred from neurons to the germline to cause transgenerational silencing.epigenetics | mobile RNAs | soma to germline | Weismann barrier

Stable epigenetic changes appear uncommon, suggesting that changes typically dissipate or are repaired. Changes that stably alter gene expression across generations presumably require particular conditions that are currently unknown. Here we report that a minimal combination of cis-regulatory sequences can support permanent RNA silencing of a single-copy transgene and its derivatives in C. elegans simply upon mating. Mating disrupts competing RNA-based mechanisms to initiate silencing that can last for >300 generations. This stable silencing requires components of the small RNA pathway and can silence homologous sequences in trans. While animals do not recover from mating-induced silencing, they often recover from and become resistant to trans silencing. Recovery is also observed in most cases when double-stranded RNA is used to silence the same coding sequence in different regulatory contexts that drive germline expression. Therefore, we propose that regulatory features can evolve to oppose permanent and potentially maladaptive responses to transient change.

Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.

Graphical Abstract:Random sets of cells can either receive different amounts of double-stranded RNA from neurons or use different RdRPs -RRF-1 only versus RRF-1 or EGO-1 -to perform the same function. ABSTRACTDelivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode C. elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissuespecific -EGO-1 in the germline and RRF-1 in somatic cells. Here we analyze instances of silencing in somatic cells that lack RRF-1. By varying dsRNA sources and target genes, we find that silencing in the absence of RRF-1 is most obvious when dsRNA from neurons is used to silence genes in intestinal cells. This silencing requires EGO-1, but the lineal identity of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. As a result, all cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against the use of a few molecules as proxies for predicting the behavior of a cell. 16: P14-P15).Transgenesis: C. elegans was transformed with plasmids and/or PCR products using microinjection (32) to generate extrachromosomal or integrated arrays. pHC337 was used to express an inverted repeat of gfp in neurons (11), which is expected to generate a hairpin RNA (gfp-dsRNA). Generation of the array that expresses unc-22-dsRNA in neurons (qtEx136) was described earlier (12). To rescue silencing defects in rde-1(jam1) and rrf-1(jam3) animals (Supplementary Figure S2), genomic DNA from wild-type animals (N2 gDNA) was used as a template to generate fused promoter/gene products through overlap extension PCR using Expand Long Template polymerase (Roche) and PCR products were purified using QIAquick PCR Purification Kit (Qiagen). The plasmid pHC448for DsRed2 expression in the pharynx or a PCR product, Prgef-1::DsRed2::unc-54 3'UTR, for DsRed2 expression in neurons was used as a co-injection marker (12). Additional details are provided in Supplementary Materials and Methods.Genome editing: Synthetic CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (IDT) or single guide RNAs (sgRNA) transcribed in vitro were combined with Cas9 protein

Gene silencing is a significant obstacle to genome engineering and has been proposed to be a non-self response against foreign DNA 1,2,3,4 . Yet, some foreign genes remain expressed for many generations 1,3,4 and some native genes remain silenced for many generations 1,5,6 . How organisms determine whether a sequence is expressed or silenced is unclear. Here we show that a stably expressed foreign DNA sequence in C. elegans is converted into a stably silenced sequence when males with the foreign DNA mate with wild-type hermaphrodites. This conversion does not occur when the hermaphrodite also has exonic sequences from the foreign DNA. Once initiated, silencing persists for many generations independent of mating and is associated with a DNA-independent signal that can silence other homologous loci in every generation. This mating-induced silencing resembles piRNA- generations. Thus, our results reveal the existence of a mechanism that maintains gene silencing initiated upon ancestral mating. By allowing retention of potentially detrimental sequences acquired through mating, this mechanism could create a reservoir of sequences that contribute to novelty when activated during evolution. ResultsMating is routinely used to introduce genes, including fluorescent reporters, into different genetic backgrounds and it is generally assumed that gene expression is unaffected by this manipulation. While