文摘
RNA interference (RNAi) plays a crucial role as an antiviral defense in several organisms including plants and invertebrates. An understanding of RNAi machineries especially protein components of the RNA-induced silencing complex (RISC) is essential for prior to applying RNAi as a tool for viral protective immunity in shrimp. Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved protein and is one of the RISC components. In previous study, suppression of Penaeus monodon TSN (PmTSN) by double-stranded RNA (dsRNA) resulted in decreasing dsRNA-mediated gene silencing activity. To elucidate the functional significance of PmTSN in shrimp RNAi pathway, interactions between PmTSN and three Argonaute proteins (PmAgo) were characterized by yeast two-hybrid and in?vitro pull-down assays. The results demonstrated that PmTSN interacted with PmAgo1, but not with PmAgo2 or PmAgo3. The interaction between PmAgo and PmTSN was mediated through the N-terminal domain of PmAgo1 and the SN1-2 domains of PmTSN. Analysis of the nuclease activity of the recombinant PmTSN indicated that PmTSN possessed calcium-dependent nuclease activity specific to single-stranded RNA (ssRNA), but not dsRNA and DNA. Knockdown of PmAgo1 and PmTSN diminished the ability of dsRNA-Rab7 to knockdown PmRab7 expression, indicating the involvement of PmAgo1 and PmTSN in shrimp RNAi pathway. Taken together, the results imply that PmTSN is one of the components of PmAgo1-RISC, thus providing new insights in the RNAi-based mechanism in shrimp.
