Michelle L. Peters, Aditi Shukla, Zoe A. Gotthold, Dylan M. McCormick, Kehui Xiang et al.
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 assembly of parasite monosomes on uAUGs, ribosomes seldom translate uORFs. These determinants of translation 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.
Michelle L. Peters, Aditi Shukla, Zoe A. Gotthold, Dylan M. McCormick, Kehui Xiang, David P. Bartel, Sebastian Lourido. 5′ untranslated regions tune Toxoplasma translation. 2025-07-14. https://doi.org/10.1101/2025.07.14.664749
A 50,000-member synthetic 5′ UTR library derived from 12 endogenous Toxoplasma UTRs was cloned upstream of mNeonGreen in an RH reporter, targeted to a neutral chromosome VI locus, and sorted by mKate-positive/mNeonGreen intensity into four bins. Two biological replicates were sequenced and author QC yielded MPRA scores for 30,235 unique sequences.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 8 files (ZIP)GSE302108_bulk_input_unique_sequences.fasta.gzGSE302108_family.soft.gzGSE302108_MPRA_score_data.txt.gzGSE302108_RAW.tarNIHPP2025.07.14.664749v1-supplement-8.pdfSupplementaryData5_MPRA_scoring.xlsxSupplementaryData6_RandomForest.xlsxSupplementaryData7_UTR_reporters_qPCR_FluorescenceIntensity.xlsx