Exogenous dsRNA silences genes in C. elegans

In 1998, antisense RNA was known to regulate gene expression in cell lines, plants and worms. It was puzzling, however, that in the nematode Caenorhabditis elegans, injection of either sense or antisense RNA resulted in transcription interference that could be transmitted to offspring. Fire, Mello and colleagues aimed to uncover some of the mysteries behind what they coined ‘RNA interference’ (RNAi). They studied the twitching phenotype caused by a reduction in the expression of the gene unc‑22; complete loss of unc‑22 expression results in more severe muscular defects and impaired motility.

To examine whether single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) contributed to the twitching effect, a 742‑nucleotide ssRNA homologous to unc‑22 was purified and its ability to silence unc‑22 relative to the homologous dsRNA was compared. Whereas the ssRNA produced only incremental effects, co‑injection of sense and antisense RNA, rather than consecutive injections, was highly effective in producing a twitching effect in the adult worm. This phenotype was heritable, although progeny were expected to maintain only a few RNA molecules per cell at the 500‑cell stage, which is when unc‑22 expression begins.

That gene silencing was likely mediated by dsRNA was further inferred by the fact that injecting the worms with gel-purified dsRNA phenocopied silencing. No effect was observed after injection of control dsRNA that was either not related to or that targeted promoter or intronic regions of unc‑22. Gene silencing effects could be reproduced by dsRNA, but not ssRNA, that was homologous to three other genes with well-characterized phenotypes. At this point, the authors deduced that dsRNA was involved, that the stoichiometry between the dsRNA and the endogenous target mRNA was not required to be 1:1, and that the response was specific to the targeted mRNA.

To visualize the silencing effect, the authors used mex‑3, a transcript that is abundant in early embryos and can be easily detected using in situ hybridization. They found that the mex‑3 transcript was not detectable following injection of dsRNA derived from mex‑3. Surprisingly, injection of purified mex‑3 antisense RNA did not significantly affect mex‑3 transcript levels. Another unexpected finding was that regardless of where in the worm dsRNA was injected, gene silencing was observed in the somatic tissue of the injected worm as well as in its progeny, suggesting the involvement of RNA transport.

Although the mechanism of RNAi remained unclear at the time, this seminal work was the first to show that strong gene silencing can be mediated by dsRNA. It laid the ground-work for a decade of studies that characterized the molecular mechanism underlying the RNAi pathway (Milestone 7) and showed that RNAi is a widespread endogenous phenomenon.