Mechanism of translation initiation in protozoan parasite Giardia lamblia
Startup Project (May 1, 2015 – April 30, 2017)
Giardia lamblia, a flagellated protozoan parasite, is the causative agent of gastrointestinal disease giardiasis in humans and is responsible for major waterborne outbreaks of diarrhea in the United States. Metronidazole has been the drug choice for treating the disease, however adverse side effects and the development of drug resistance strains have necessitated the search for new drugs. Preliminary observations have indicated that Giardia mRNAs have very short 5’UTRs and that translation initiation in Giardia occurs via direct ribosome binding, without the elaborate transcript scanning process that is typical in higher eukaryotes. In yeast and mammals, translation initiation factor eIF4G forms a trimeric complex eIF4F by binding with two other proteins, a 5’ cap-binding protein eIF4E and a ATP-dependent RNA helicase eIF4A and eIF4G is a key player in binding pre- initiation complexes to 5’end of mRNA. However, homology based searches of Giardia genome database have identified eIF4E and eIF4A homologs but failed to identify a homolog for eIF4G. It is not clear as to how pre-initiation complexes are recruited to the mRNA in the absence of a key initiation factor eIF4G. Since, GleIF4E2, a cap-binding homolog of eIF4E, binds in close proximity to the initiation codon, it is hypothesized that GleIF4E2 interacts with the components of the pre-initiation complex and facilitates direct binding of the pre-initiation complex to initiation codon. We will use two complementary approaches to identify the GleIF4E2 interacting proteins in the pre-initiaition complex. The proteins will be purified by these two approaches will be identified by mass spectrometry. The interaction of GleIF4E2 with identified proteins will be validated by co-immunoprecipitation, GST-pull down assays and yeast-two hybrid analysis. Characterizing the GleIF4E2 interacting proteins will help us understand the process of translation initiation in Giardia. Although scanning mechanism is absent in Giardia, presence of eIF4A homolog GleIF4A in Giardia suggests that it has a role in translation initiation. We will investigate the role of GleIF4A in translation by knocking down the gene expression and using eIF4A inhibitors hippuristionl and pateamine A.