Characterizing the impact of mRNA regulatory features on translation efficiency in Toxoplasma gondii

Protein expression can be regulated through cis and trans factors acting on mRNA and translation machinery. This regulation allows cells to fine-tune expression of specific factors in response to environmental cues. The transcriptome of Toxoplasma gondii represents a challenge to canonical models of translational regulation in eukaryotes as its 5′ UTRs are several times longer than most other characterized species and the vast majority of 5′ UTRs harbor at least one, if not several, upstream AUGs. We hypothesize that these unusual features may be maintained in Toxoplasma because they confer regulatory information important for gene expression across the parasite life cycle. To understand how the parasite regulates translation across both its tachyzoite and bradyzoite stages, we performed high-resolution ribosome profiling on Toxoplasma and human host cells. Bioinformatic analyses of these data have characterized general transcript features that contribute to translation efficiency differently in Toxoplasma and human cells. Additionally, we identified a cohort of translationally-regulated factors whose expression changes between tachyzoites and bradyzoites. Based on these data, we aim to dissect the relative contributions of mRNA regulatory features to translation efficiency in the parasite. Using quantitative reporter systems, we are examining the sufficiency of endogenous 5′ UTRs to regulate both steady-state protein expression and changes under stress. Mutational analyses will reveal how specific sequence features, such as upstream open reading frames, impact translation efficiency. Together, this work expands our molecular framework for gene regulation in Toxoplasma.