Extracellular RNAs: Directing nuclear and cytoplasmic silencing from the pseudocoelom.

摘要

The experimental introduction of dsRNA within a eukaryotic cell results in the down regulation of genes in a sequence specific fashion. This gene inhibition, known as RNAi, is one of several pathways in which cells use non-coding RNAs to provide specificity in gene regulatory processes. Interestingly, in some animals RNAi results in a systemic response where gene silencing spreads throughout the animal from the site of initial dsRNA exposure. In the nematode C. elegans the identity of some of the transporters involved in the movement of the silencing signals are known, although the molecular identity and path taken by the silencing signal itself is still a mystery. Also unknown is whether any of the other RNA mediated gene regulatory processes use the systemic RNAi machinery to facilitate intercellular gene regulation. To answer these questions I developed a technique to test the extracellular space for silencing signals during RNAi responses. These experiments demonstrate that silencing signals do indeed travel extracellularly, and are primarily composed of dsRNAs that feed into the RNAi pathway above RDE-4, which binds long dsRNAs. Additionally, the process of RNAi appears to be regulated to ensure a tempered response to trigger dsRNA by simultaneously minimizing signal amplification as well as maintaining downstream silencing molecules as cell-autonomous. Interestingly, one of these downstream processes, transitive RNAi, is enhanced upon disruption of the endogenous RNAi pathway. I have characterized this enhancement and found it to require the nuclear argonaute NRDE-3. This requirement is not unique to transitivity, as enhanced transgene silencing and RNAi targeting gene families also require NRDE-3. Additional work also shows that several of the exogenous RNAi pathway genes play a role in endogenous RNAi.