Small interfering RNAs (siRNAs) mediate gene silencing by basepairing with their target RNA sequences. While near perfect complementarity between siRNAs and their target RNA sequences is required for efficient gene silencing, exposure of eukaryotes to siRNAs frequently results in unintended silencing of genes exhibiting < 100%sequence identity to the trigger siRNAs; a process termed RNAi off-target effect. RNAi off-target effect is one of the major concerns for the application of RNAi-based technology in human therapeutics. We developed a method to study the RNAi off-target effect inC. elegans.
dpy-13 is a collagen gene, which belongs to a large gene familyt that contains more than 150 members sharing high sequence similarity. Animals harboring a null allele
dpy-13(
e458) exhibit a dumpy phenotype. Exposure of wild type N2 animals to dsRNA targeting
dpy-13 induces a dumpy phenotype less severe than the
dpy-13 null mutant. eri(-)animals exhibit enhanced sensitivity to RNAi. Feeding eri(-) animals with dpy-13dsRNA elicits a phenotype which is extremely more severe (super-dumpy) than that of
dpy-13 null animals. In the
dpy-13(
e458) strain, a strain deleting most of the gene, however, feeding dsRNA targeting this deleted region still results in a super-dumpy phenotype. Thus, we postulate that dsRNA targeting the
dpy-13 gene is able to trigger an RNAioff-target effect by silencing nucleic acid sequences outside of the
dpy-13 locus; possibly other collagens with high sequence similarity. We examined the genetic requirements for this RNAi off-target effect. nrde genes are necessary to silence nuclear localized RNAs. In nrde(-)animals, silencing of the nuclear localized RNAs is defective, while the cytoplasmic RNAi pathway remains functional. Interestingly, nrde(-) animalsare resistant to the
dpy-13 RNAi induced super-dumpy phenotype. NRDE-3 is an Argonaute protein and it specifically associates with its target pre-mRNA after RNAi. We found that NRDE-3 interacts with
dpy-13,
sqt-3 and
col-43 sequences after dpy-13RNAi. Moreover, targeting the
sqt-3gene by RNAi in
dpy-13(
e458) mutant animals elicits a super-dumpy phenotype. This supports the idea that dpy-13RNAi likely results in off-target silencing of other collagen genes throughnrde. Thus, unlike the conventional view that RNAi off-targeting isan unavoidable phenomenon, our data suggest at least some of these effects are intimately dependent on the nuclear RNAi pathway.