RISC in Entamoeba histolytica: Identification of a Protein-Protein Interaction Network for the RNA Interference Pathway in a Deep-Branching Eukaryote.

Entamoeba histolytica is a protozoan parasite that causes amebiasis in humans and is a major health concern in developing countries. Our previous work revealed a functional RNA interference (RNAi) pathway in . Several unusual features encompass the RNAi pathway in the parasite, including small RNAs (sRNAs) with a 5'-polyphosphate structure (identified to date only in and nematodes) and the conspicuous absence of a canonical Dicer enzyme. Currently, little is known about the RNA-induced silencing complex (RISC), which is critical in understanding how RNAi is achieved in the parasite. In this study, we examined the RISC of Ago2-2, the most highly expressed Argonaute protein in . We identified 43 protein components of Ago2-2 RISC with a broad range of functional activities. Two proteins with nucleosome assembly protein (NAP) domains, not previously observed in other RNAi systems, were identified as novel core members of amebic RISC. We further demonstrated the interaction of these NAPs with Ago using an recombinant system. Finally, we characterized the interaction network of five RISC components identified in this study to further elucidate the interactions of these RNAi pathway proteins. Our data suggest the presence of closely interacting protein groups within RISC and allowed us to build a map of protein-protein interactions in relation to Ago. Our work is the first to elucidate RISC components in and expands the current knowledge of RISC to a deep-branching single-celled eukaryote. Entamoeba histolytica is a leading parasitic cause of death in developing countries, and our efforts are focused on defining the molecular basis of RNA interference (RNAi) gene regulation in this parasite. The RNAi pathway effectively silences a subset of endogenous genes and has also been harnessed as a gene silencing tool to study gene function in this organism. However, little is known about the components of the RNA-induced silencing complex (RISC), which is critical in understanding how gene silencing is achieved in the parasite. This study characterizes, for the first time, the RISC components in and provides new insights in understanding the molecular regulatory mechanisms of RNAi in this parasite, including the demonstration of novel Ago protein-interacting partners. From an evolutionary point of view, our findings expand the current knowledge of RISC to a deep-branching single-celled eukaryote.