5' Untranslated regions tune Toxoplasma translation [MPRA]

Some of the longest 5′ untranslated regions (UTRs) documented in eukaryotes belong to parasites of the phylum Apicomplexa. Translational regulation plays prominent roles in the development of these parasites, including the agents of toxoplasmosis (Toxoplasma gondii) and malaria. To understand the function of 5′ UTRs in apicomplexan translation, we performed high-resolution ribosome profiling of T. gondii in human fibroblasts. We show that parasite translation efficiency (TE) is largely controlled by 5′ UTR features and tuned by the number of upstream AUGs (uAUGs). Examination of ribosome occupancy reveals that, despite widespread engagement of uAUGs, parasite ribosomes seldom translate uORFs. These features are reaffirmed in a massively parallel reporter assay examining the effect of more than 30,000 synthetic 5′ UTRs in T. gondii. A model trained on these results accurately predicted the TE of newly designed 5′ UTRs. Together, this work defines the regulatory language of T. gondii translation, providing a framework to understand the evolution of exceptionally long 5′ UTRs in apicomplexans. Massively parallel reporter assay (MPRA) of 5' UTRs driving mNeonGreen, with a constitutive mKate marker. Reporter construct was stably integrated into Toxoplasma gondii (RH) and parasites were subjected to FACS based on mKate positivity and mNG intensity. Targeted DNA and RNA sequencing was performed on the plasmid library, bulk parasite, populations and 4 sorted bins.