Institute of Molecular Biosciences, Mahidol University (Salaya Campus), 25/25 Phutthamonthon 4 Road, Salaya, Phutthamonthon District, Nakhon Pathom 73170, Thailand.
Fish Shellfish Immunol. 2013 Mar;34(3):875-84. doi: 10.1016/j.fsi.2012.12.012. Epub 2013 Jan 18.
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.
RNA 干扰 (RNAi) 在包括植物和无脊椎动物在内的几种生物中作为抗病毒防御发挥着至关重要的作用。在应用 RNAi 作为虾类病毒保护性免疫的工具之前，了解 RNAi 机制，特别是 RNA 诱导沉默复合物 (RISC) 的蛋白质成分是必不可少的。结构域特异性核酸内切酶（Tudor staphylococcal nuclease，TSN）是一种进化上保守的蛋白质，是 RISC 的组成部分之一。在之前的研究中，双链 RNA（dsRNA）对凡纳滨对虾 TSN（PmTSN）的抑制导致 dsRNA 介导的基因沉默活性降低。为了阐明 PmTSN 在虾类 RNAi 途径中的功能意义，通过酵母双杂交和体外下拉实验研究了 PmTSN 与三种 Argonaute 蛋白（PmAgo）之间的相互作用。结果表明，PmTSN 与 PmAgo1 相互作用，但不与 PmAgo2 或 PmAgo3 相互作用。PmAgo 与 PmTSN 的相互作用是通过 PmAgo1 的 N 端结构域和 PmTSN 的 SN1-2 结构域介导的。重组 PmTSN 的核酸酶活性分析表明，PmTSN 具有钙依赖性的单链 RNA（ssRNA）特异性核酸酶活性，但不具有 dsRNA 和 DNA 特异性核酸酶活性。PmAgo1 和 PmTSN 的敲低降低了 dsRNA-Rab7 敲低 PmRab7 表达的能力，表明 PmAgo1 和 PmTSN 参与了虾类 RNAi 途径。总之，这些结果表明 PmTSN 是 PmAgo1-RISC 的组成部分之一，从而为虾类基于 RNAi 的机制提供了新的见解。
