Identification and characterization of an atypical RIG-I encoded by planarian Dugesia japonica and its essential role in the immune response.

Retinoic acid-inducible gene I (RIG-I), an RNA sensor with a conserved structure, activates the host interferon (IFN) system to produce IFNs and cytokines for eliminating pathogens upon recognizing PAMPs. However, the biological functions and the mechanism by which RIG-I regulates the innate immunity response in invertebrates are still unknown at present. Here we identified an atypical RIG-I in planarian Dugesia japonica. Sequence analysis, 3D structure modeling and phylogenetic analysis showed that this atypical protein was clustered into a single clade at the base of the tree in invertebrates, suggesting that DjRIG-I is an ancient and unique protein of the RIG-I-like receptors (RLRs). In situ hybridization analysis revealed that the DjRIG-I mRNAs were predominantly expressed in the pharynx and head of the adult and regenerative planarians. Stimulation with PAMPs induced the over-expression of DjRIG-I in planarians. The molecular simulation demonstrated that DjRIG-I formed a large hole-structure for the docking of dsRNAs, and the pull-down assay confirmed the interaction between DjRIG-I and viral analog poly(I:C). Importantly, some representative antiviral/antibacterial genes in the RIG-I-mediated IFN and P38 signaling pathway, TBK1, IRF-3, Mx, and P38, were significantly upregulated in planarians stimulated with PAMPs. Interference of the DjRIG-I expression by RNAi, inhibited the PAMPs-induced over-expression, suggesting that DjRIG-I is a key player for downstream signaling events. These results indicate that DjRIG-I triggered the intracellular signaling cascades independent of the classical CARD domains and played an essential role in the virus/bacteria-induced innate immunity of planarian.