State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China.
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China.
Sci Rep. 2017 Mar 20;7:44832. doi: 10.1038/srep44832.
RNaseIII proteins are dsRNA-specific endonucleases involved in many important biological processes, such as small RNA processing and maturation in eukaryotes. Various small RNAs have been identified in a protozoan parasite Entamoeba histolytica. EhRNaseIII is the only RNaseIII endonuclease domain (RIIID)-containing protein in E. histolytica. Here, we present three crystal structures that reveal several unique structural features of EhRNaseIII, especially the interactions between the two helixes (α1 and α7) flanking the RIIID core domain. Structure and sequence analysis indicate that EhRNaseIII is a noncanonical Dicer and it lacks a dsRBD in the C-terminal region (CTR). In vitro studies suggest that EhRNaseIII prefers to bind and cleave longer dsRNAs, generating products around 25 nucleotides in length. Truncation of the CTR or attaching the dsRBD of Aquifex aeolicus RNaseIII can enhance the binding and cleavage activities of EhRNaseIII. In combination with in vitro crosslinking assay, our results suggested that EhRNaseIII functions in a cooperative mode. We speculate that some partner proteins may exist in E. histolytica and regulates the activity of EhRNaseIII through interaction with its CTR. Our studies support that EhRNaseIII plays an important role in producing small RNAs in E. histolytica.
RNaseIII 蛋白是 dsRNA 特异性内切核酸酶，参与许多重要的生物学过程，如真核生物中小 RNA 的加工和成熟。在原生动物寄生虫溶组织内阿米巴中已经鉴定出各种小 RNA。EhRNaseIII 是溶组织内阿米巴中唯一含有 RNaseIII 内切酶结构域（RIIID）的蛋白。在这里，我们呈现了三个晶体结构，揭示了 EhRNaseIII 的几个独特结构特征，特别是 RIIID 核心结构域侧翼的两个螺旋（α1 和 α7）之间的相互作用。结构和序列分析表明，EhRNaseIII 是一种非典型的 Dicer，它在 C 端区域（CTR）缺乏 dsRBD。体外研究表明，EhRNaseIII 更喜欢结合和切割较长的 dsRNA，产生大约 25 个核苷酸长的产物。CTR 的截断或附着 Aquifex aeolicus RNaseIII 的 dsRBD 可以增强 EhRNaseIII 的结合和切割活性。结合体外交联实验，我们的结果表明 EhRNaseIII 以协同模式发挥作用。我们推测在溶组织内阿米巴中可能存在一些伴侣蛋白，通过与 EhRNaseIII 的 CTR 相互作用来调节其活性。我们的研究支持 EhRNaseIII 在产生溶组织内阿米巴中小 RNA 中发挥重要作用。
