טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAbed Mona
SubjectApplications of RNAi in the Protozoan Parasite Entamoeba
histolytica
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri


Abstract

Amebiasis, an intestinal disease caused by a protozoan parasite called Entamoeba histolytica (E.histolytica), is considered the second leading cause of death from parasitic disease worldwide. Elucidation of the genes that determine the nature of the relationship between the Entamoeba histolytica and its host are of great interest. A common way to determine a gene function is to inactivate the gene, and then examine the resulting phenotype. Yet several features of E.histolytica, like their variable DNA content and complex ploidity have made it difficult to perform classical genetic studies like homologous recombination. RNA interference is a mechanism through which dsRNA molecules corresponding to target gene sequences silence cognate genes. In this study we identify and partially characterize two proteins involved in the RNA interference (RNAi) pathway, RNase III and AGO2. RNaseIII is the first enzyme in the RNAi pathway which triggers gene inactivation by cleaving dsRNA corresponding to the target gene into smaller dsRNA fragments called siRNA. We demonstrate that E.histolytica expresses a homolog to the bacterial RNaseIII, which when expressed as a recombinant protein can cleave dsRNA. After dsRNA is cleaved into siRNA, the short fragments act as guide RNA which lead an enzyme complex called RISC to the corresponding mRNA. Once bound, the RISC cleaves the mRNA which results in its further degradation by cellular nucleases. We found that AGO, the only protein within the RISC complex to be conserved between organisms is also expressed in E.histolytica. Our results strengthen the findings that an RNAi pathway does exist in this parasite. With these findings we set out to inactivate several E.histolytica genes by various dsRNA delivery methods described in other eukaryotic cells.