Characterization of an RNase III protein and its potential roles in the RNA interference pathway of the protozoan parasite, Entamoeba histolytica [electronic resource]

Entamoeba histolytica, a human intestinal parasite and a leading cause of death worldwide, contains a complex repertoire of endogenous small RNAs. Core elements of the RNAi machinery have been identified in the E. histolytica genome including three Argonaute proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) and two genes with RNA-directed RNA polymerase (RdRP) domains. However, to date no canonical Dicer enzyme, an RNaseIII endonuclease responsible for cleaving double-stranded RNA (dsRNA) to 20-30nt small RNAs and a critical player in the RNAi pathway, has been identified in the E. histolytica genome. We conducted bioinformatics searches of the genome which revealed only one gene with an RNaseIII domain, EhRNaseIII. Interestingly, this candidate lacks the canonical Dicer structure - it is substantially smaller than other Dicers, contains only a single RNaseIII domain, and lacks PAZ or double-stranded RNA binding domain (dsRBD). Most eukaryotic Dicer proteins identified to date contain two RNaseIII domains, which form a heterodimer required for cleavage of the dsRNA, and contain PAZ or dsRBDs. We determined that EhRNaseIII exists as a homodimer in E. histolytica trophozoites. We sought to probe the contributions of EhRNaseIII, EhAgo2-2, and EhRdRP1 to the RNAi pathway in E. histolytica. Attempts to downregulate these genes using RNA-based methods including antisense mediated silencing and the production of gene-specific secondary small RNAs were unsuccessful. Expression of EhRNaseIII dominant-negative mutants also failed to impair production of small RNAs. In order to determine if EhRNaseIII was capable of generating small RNAs of the size observed in E. histolytica trophozoites, we conducted in vitro cleavage assays of dsRNA. Despite using multiple protein sources (E. histolytica whole cell lysate, immunoprecipitated EhRNaseIII, and recombinant EhRNaseIII protein) and two structurally distinct dsRNA substrates, we were unable to detect small RNA cleavage products. We further investigated whether EhRNaseIII was sufficient to act as a Dicer enzyme by co-expressing it with a dsRNA substrate in Saccharomyces cerevisiae, which lacks an RNAi pathway. However, no small RNAs were detected. We also assessed whether EhAgo2-2, which associates with 5'-polyphosphate small RNAs in E. histolytica, could mediate silencing using 5'-monophosphated small RNAs in S. cerevisiae. No downregulation in reporter transcript or protein was observed. These data indicate that EhRNaseIII is not a minimal Dicer enzyme and suggest that small RNA biogenesis may occur solely through a Dicer-independent small pathway in E. histolytica. These data also suggest that EhAgo2-2 may be specific for 5'-polyphosphate small RNAs. Future studies will continue to investigate the mechanism of small RNA-induced silencing in E. histolytica.

Keywords: Microbiology and Immunology.
Advisor: Singh Upinder (Physician) primary advisor. Thesis advisor
Boothroyd John C. advisor. Thesis advisor
Chen Chang-Zheng advisor. Thesis advisor
Fire Andrew Zachary advisor. Thesis advisor