Constructing a Dual Luciferase Reporter Assay System to Understand Translational Regulation in Plasmodium falciparum
Authors:
Vida Ahyong, Jonathan DiepMentor:
Joseph DeRisi, Professor of Biochemistry and Biophysics, University of California San FranciscoThe protozoan parasite Plasmodium falciparum causes the most acute form of human malaria. The development of new antimalarials against multidrug-resistant P. falciparum, however, has been stymied by the lack of knowledge about many aspects of its basic biology. Ribosome profiling conducted in our lab revealed that mRNAs present during the schizont stage of the erythrocyte cycle have the lowest translation efficiency and a high density of ribosome binding to upstream open reading frames (uORFs) in the 5’ untranslated region (UTR). To determine whether ribosome occupancy on uORFs has an effect on translational efficiency, we chose to focus on EBA-175, a well-characterized erythrocyte binding protein of P. falciparum. A mutant 5’ UTR that changes the AUG start codons to UAG stop codons for all uORFs was synthesized. By eliminating the putative AUG ribosome binding sites for the uORFs in the 5’UTR, we hypothesize that translational downregulation of EBA-175 during the schizont stage will be relieved. To test this hypothesis, a dual luciferease reporter assay system was constructed to compare downstream firefly and renilla luciferase production in vitro between the wild-type and mutant 5’ UTR of EBA-175. The wild-type and mutant luciferase reporter constructs were generated by fusion polymerase chain reaction, and their functionality was verified by in vitro translation in rabbit reticulocyte with millions of luminescence units above the negative background. At the moment, we are working towards optimizing conditions for in vitro translation in cell-free extracts of P. falciparum. By understanding the mechanism of translational regulation for P. falciparum, we will be able to identify novel pathways in the erythrocyte cycle that could be used as future drug targets for antimalarials.