Gene silencing by double-stranded RNA fromC. elegansneurons reveals functional mosaicism of RNA interference

Abstract

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

Intergenerational transport of double-stranded RNA inC. eleganscan limit heritable epigenetic changes

“…These temporal and/or spatial preferences for silencing could be because of unknown characteristics of the exported neuronal dsRNA (e.g., modifications, lengths, structures, etc.) that influence import or subsequent silencing -a hypothesis that is also supported by the different requirements for silencing by neuronal gfp-dsRNA compared to other sources of gfp-dsRNA (21). Alternatively, these preferences could reflect universal constraints for any extracellular dsRNA in C. elegans.…”

“…Secretion of dsRNA from C. elegans tissues that express dsRNA has been inferred based upon the SID-1-dependent silencing of matching genes in other tissues (13,20). Secreted dsRNA from neurons can silence genes of matching sequence in most somatic cells (21) and within the germline (22). Extracellular dsRNA delivered into parental circulation by injection or ingestion also enters the germline and can cause silencing of matching genes in progeny (12,(23)(24)(25)(26).…”

A framework for parsing heritable information

“…However because there are two possible cases (upper vs. lower), there would be two dis-tinguishable states that are effectively equivalent for survival in this environment. Such situations could result in unregulated redundancy such that similar functions are performed by different molecules in random sets of cells [33]. Over evolutionary time scales, this type of unregulated redundancy could result in organisms with similar form and function but different underlying molecular mechanisms [34].…”