{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:58:56Z","timestamp":1785430736821,"version":"3.56.0"},"posted":{"date-parts":[[2025,7,14]]},"group-title":"Molecular Biology","reference-count":86,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,14]],"date-time":"2025-07-14T00:00:00Z","timestamp":1752451200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["AI144369"],"award-info":[{"award-number":["AI144369"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["AI158501"],"award-info":[{"award-number":["AI158501"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/01d35cw23","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/037ebw447","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,14]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Some of the longest 5\u2032 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 (\n                  <jats:italic>Toxoplasma gondii<\/jats:italic>\n                  ) and malaria. To understand the function of 5\u2032 UTRs in apicomplexan translation, we performed high-resolution ribosome profiling of\n                  <jats:italic>T. gondii<\/jats:italic>\n                  in human fibroblasts. We show that parasite translation efficiency (TE) is largely controlled by 5\u2032 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\u2032 UTRs in\n                  <jats:italic>T. gondii<\/jats:italic>\n                  . A model trained on these results accurately predicted the TE of newly designed 5\u2032 UTRs. Together, this work defines the regulatory language of\n                  <jats:italic>T. gondii<\/jats:italic>\n                  translation, providing a framework to understand the evolution of exceptionally long 5\u2032 UTRs in apicomplexans.\n                <\/jats:p>","DOI":"10.1101\/2025.07.14.664749","type":"posted-content","created":{"date-parts":[[2025,7,15]],"date-time":"2025-07-15T04:50:10Z","timestamp":1752555010000},"source":"Crossref","is-referenced-by-count":0,"title":["5\u2032 untranslated regions tune\n                  <i>Toxoplasma<\/i>\n                  translation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2919-2523","authenticated-orcid":false,"given":"Michelle L.","family":"Peters","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9743-4128","authenticated-orcid":false,"given":"Aditi","family":"Shukla","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-7092-2775","authenticated-orcid":false,"given":"Zoe A.","family":"Gotthold","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3373-9891","authenticated-orcid":false,"given":"Dylan M.","family":"McCormick","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3770-1367","authenticated-orcid":false,"given":"Kehui","family":"Xiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3872-2856","authenticated-orcid":false,"given":"David P.","family":"Bartel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5237-1095","authenticated-orcid":false,"given":"Sebastian","family":"Lourido","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025071600050591000_2025.07.14.664749v1.1","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a011528"},{"key":"2025071600050591000_2025.07.14.664749v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2009.01.042"},{"key":"2025071600050591000_2025.07.14.664749v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-023-00624-9"},{"key":"2025071600050591000_2025.07.14.664749v1.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.aad9868"},{"key":"2025071600050591000_2025.07.14.664749v1.5","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0810916106"},{"key":"2025071600050591000_2025.07.14.664749v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21394-y"},{"key":"2025071600050591000_2025.07.14.664749v1.7","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.00136-12"},{"key":"2025071600050591000_2025.07.14.664749v1.8","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.200408003"},{"key":"2025071600050591000_2025.07.14.664749v1.9","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.59.031805.133833"},{"key":"2025071600050591000_2025.07.14.664749v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.gene.2004.11.041"},{"key":"2025071600050591000_2025.07.14.664749v1.11","doi-asserted-by":"crossref","unstructured":"Lawless, C. et al. Upstream sequence elements direct post-transcriptional regulation of gene expression under stress conditions in yeast. BMC Genomics 10, 7 (2009).","DOI":"10.1186\/1471-2164-10-7"},{"key":"2025071600050591000_2025.07.14.664749v1.12","doi-asserted-by":"crossref","unstructured":"Markus, B. M. , Waldman, B. S. , Lorenzi, H. A. & Lourido, S . High-resolution mapping of transcription initiation in the asexual stages of Toxoplasma gondii. Front. Cell. Infect. Microbiol. 10, 617998 (2020).","DOI":"10.3389\/fcimb.2020.617998"},{"key":"2025071600050591000_2025.07.14.664749v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2016.02.025"},{"key":"2025071600050591000_2025.07.14.664749v1.14","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1002\/wrna.1365","article-title":"Translational regulation in blood stages of the malaria parasite Plasmodium spp.: systems-wide studies pave the way","volume":"7","year":"2016","journal-title":"Wiley Interdiscip. Rev. RNA"},{"key":"2025071600050591000_2025.07.14.664749v1.15","doi-asserted-by":"publisher","DOI":"10.1126\/science.1125129"},{"key":"2025071600050591000_2025.07.14.664749v1.16","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002046"},{"key":"2025071600050591000_2025.07.14.664749v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2019.12.013"},{"key":"2025071600050591000_2025.07.14.664749v1.18","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1038\/s41564-023-01358-2","article-title":"A positive feedback loop controls Toxoplasma chronic differentiation","volume":"8","year":"2023","journal-title":"Nat. Microbiol"},{"key":"2025071600050591000_2025.07.14.664749v1.19","doi-asserted-by":"crossref","first-page":"4385","DOI":"10.1038\/s41467-024-48685-4","article-title":"Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors","volume":"15","year":"2024","journal-title":"Nat. Commun"},{"key":"2025071600050591000_2025.07.14.664749v1.20","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1121567109"},{"key":"2025071600050591000_2025.07.14.664749v1.21","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20091975"},{"key":"2025071600050591000_2025.07.14.664749v1.22","doi-asserted-by":"publisher","DOI":"10.1042\/bj20040262"},{"key":"2025071600050591000_2025.07.14.664749v1.23","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02954-23"},{"key":"2025071600050591000_2025.07.14.664749v1.24","doi-asserted-by":"crossref","first-page":"e1009816","DOI":"10.1371\/journal.ppat.1009816","article-title":"Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii","volume":"17","year":"2021","journal-title":"PLoS Pathog"},{"key":"2025071600050591000_2025.07.14.664749v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/nrm4069"},{"key":"2025071600050591000_2025.07.14.664749v1.26","doi-asserted-by":"publisher","DOI":"10.1126\/science.1168978"},{"key":"2025071600050591000_2025.07.14.664749v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2016.01.043"},{"key":"2025071600050591000_2025.07.14.664749v1.28","doi-asserted-by":"crossref","unstructured":"Holmes, M. J. , Shah, P. , Wek, R. C. & Sullivan, W. J., Jr . Simultaneous ribosome profiling of human host cells infected with Toxoplasma gondii. mSphere 4, (2019).","DOI":"10.1128\/mSphere.00292-19"},{"key":"2025071600050591000_2025.07.14.664749v1.29","doi-asserted-by":"crossref","unstructured":"Hassan, M. A. , Vasquez, J. J. , Guo-Liang, C. , Meissner, M. & Siegel, T. N . Comparative ribosome profiling uncovers a dominant role for translational control in Toxoplasma gondii. BMC Genomics 18, 961 (2017).","DOI":"10.1186\/s12864-017-4362-6"},{"key":"2025071600050591000_2025.07.14.664749v1.30","doi-asserted-by":"publisher","DOI":"10.1002\/cpmc.26"},{"key":"2025071600050591000_2025.07.14.664749v1.31","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms8816"},{"key":"2025071600050591000_2025.07.14.664749v1.32","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad1003"},{"key":"2025071600050591000_2025.07.14.664749v1.33","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0124473"},{"key":"2025071600050591000_2025.07.14.664749v1.34","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2011.10.002"},{"key":"2025071600050591000_2025.07.14.664749v1.35","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btz878"},{"key":"2025071600050591000_2025.07.14.664749v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2020.09.011"},{"key":"2025071600050591000_2025.07.14.664749v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2016.08.019"},{"key":"2025071600050591000_2025.07.14.664749v1.38","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms10147"},{"key":"2025071600050591000_2025.07.14.664749v1.39","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1002866"},{"key":"2025071600050591000_2025.07.14.664749v1.40","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.05.049"},{"key":"2025071600050591000_2025.07.14.664749v1.41","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.16.1.146"},{"key":"2025071600050591000_2025.07.14.664749v1.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.devcel.2024.12.038"},{"key":"2025071600050591000_2025.07.14.664749v1.43","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(86)90762-2"},{"key":"2025071600050591000_2025.07.14.664749v1.44","doi-asserted-by":"publisher","DOI":"10.1007\/s004360050254"},{"key":"2025071600050591000_2025.07.14.664749v1.45","doi-asserted-by":"crossref","unstructured":"Gleason, A. C. , Ghadge, G. , Sonobe, Y. & Roos, R. P . Kozak Similarity Score algorithm identifies alternative translation initiation codons implicated in cancers. Int. J. Mol. Sci. 23, 10564 (2022).","DOI":"10.3390\/ijms231810564"},{"key":"2025071600050591000_2025.07.14.664749v1.46","doi-asserted-by":"publisher","DOI":"10.1016\/j.biocel.2013.04.020"},{"key":"2025071600050591000_2025.07.14.664749v1.47","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msi100"},{"key":"2025071600050591000_2025.07.14.664749v1.48","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1207846109"},{"key":"2025071600050591000_2025.07.14.664749v1.49","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0164-5"},{"key":"2025071600050591000_2025.07.14.664749v1.50","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-24436-7"},{"key":"2025071600050591000_2025.07.14.664749v1.51","doi-asserted-by":"publisher","DOI":"10.1101\/gr.224964.117"},{"key":"2025071600050591000_2025.07.14.664749v1.52","doi-asserted-by":"crossref","unstructured":"Markus, B. M. , Bell, G. W. , Lorenzi, H. A. & Lourido, S . Optimizing Systems for Cas9 Expression in Toxoplasma gondii. mSphere 4, (2019).","DOI":"10.1128\/mSphere.00386-19"},{"key":"2025071600050591000_2025.07.14.664749v1.53","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btab203%J"},{"key":"2025071600050591000_2025.07.14.664749v1.54","unstructured":"Greenwell, B. Fastshap: Fast Approximate Shapley Values in R. (Github)."},{"key":"2025071600050591000_2025.07.14.664749v1.55","doi-asserted-by":"publisher","DOI":"10.18637\/jss.v053.i04"},{"key":"2025071600050591000_2025.07.14.664749v1.56","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2019.03.002"},{"key":"2025071600050591000_2025.07.14.664749v1.57","doi-asserted-by":"publisher","DOI":"10.1016\/S0020-7519(02)00096-6"},{"key":"2025071600050591000_2025.07.14.664749v1.58","doi-asserted-by":"crossref","first-page":"e39273","DOI":"10.7554\/eLife.39273","article-title":"Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition","volume":"7","year":"2018","journal-title":"Elife"},{"key":"2025071600050591000_2025.07.14.664749v1.59","doi-asserted-by":"publisher","DOI":"10.1101\/2024.07.10.602410"},{"key":"2025071600050591000_2025.07.14.664749v1.60","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1561-0"},{"key":"2025071600050591000_2025.07.14.664749v1.61","doi-asserted-by":"publisher","DOI":"10.1017\/S0031182020000840"},{"key":"2025071600050591000_2025.07.14.664749v1.62","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2013-14-11-r128"},{"key":"2025071600050591000_2025.07.14.664749v1.63","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.04106"},{"key":"2025071600050591000_2025.07.14.664749v1.64","doi-asserted-by":"publisher","DOI":"10.1101\/2024.08.11.607362"},{"key":"2025071600050591000_2025.07.14.664749v1.65","doi-asserted-by":"crossref","first-page":"10898","DOI":"10.1038\/s41467-024-55274-y","article-title":"NaP-TRAP reveals the regulatory grammar in 5\u2019UTR-mediated translation regulation during zebrafish development","volume":"15","year":"2024","journal-title":"Nat. Commun"},{"key":"2025071600050591000_2025.07.14.664749v1.66","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2024.11.030"},{"key":"2025071600050591000_2025.07.14.664749v1.67","doi-asserted-by":"crossref","unstructured":"Garcia, V. E. , Dial, R. & DeRisi, J. L . Functional characterization of 5\u2019 UTR cis-acting sequence elements that modulate translational efficiency in Plasmodium falciparum and humans. Malar. J. 21, 15 (2022).","DOI":"10.1186\/s12936-021-04024-2"},{"key":"2025071600050591000_2025.07.14.664749v1.68","doi-asserted-by":"crossref","unstructured":"Kornienko, I. V. , Aramova, O. Y. , Tishchenko, A. A. , Rudoy, D. V. & Chikindas, M. L . RNA stability: A review of the role of structural features and environmental conditions. Molecules 29, (2024).","DOI":"10.3390\/molecules29245978"},{"key":"2025071600050591000_2025.07.14.664749v1.69","doi-asserted-by":"publisher","DOI":"10.1261\/rna.062794.117"},{"key":"2025071600050591000_2025.07.14.664749v1.70","doi-asserted-by":"publisher","DOI":"10.7554\/elife.66493"},{"key":"2025071600050591000_2025.07.14.664749v1.71","doi-asserted-by":"publisher","DOI":"10.1038\/nature13007"},{"key":"2025071600050591000_2025.07.14.664749v1.72","doi-asserted-by":"crossref","unstructured":"Martin, M . Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10 (2011).","DOI":"10.14806\/ej.17.1.200"},{"key":"2025071600050591000_2025.07.14.664749v1.73","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts635"},{"key":"2025071600050591000_2025.07.14.664749v1.74","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw257"},{"key":"2025071600050591000_2025.07.14.664749v1.75","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1754"},{"key":"2025071600050591000_2025.07.14.664749v1.76","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btq033"},{"key":"2025071600050591000_2025.07.14.664749v1.77","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae974"},{"key":"2025071600050591000_2025.07.14.664749v1.78","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btt656"},{"key":"2025071600050591000_2025.07.14.664749v1.79","doi-asserted-by":"crossref","unstructured":"Love, M. I. , Huber, W. & Anders, S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).","DOI":"10.1186\/s13059-014-0550-8"},{"key":"2025071600050591000_2025.07.14.664749v1.80","doi-asserted-by":"crossref","first-page":"868","DOI":"10.1038\/s41564-022-01104-0","article-title":"Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress","volume":"7","year":"2022","journal-title":"Nat. Microbiol"},{"key":"2025071600050591000_2025.07.14.664749v1.81","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M115.700518"},{"key":"2025071600050591000_2025.07.14.664749v1.82","doi-asserted-by":"crossref","unstructured":"Herneisen, A. L. , Peters, M. L. , Smith, T. A. , Shortt, E. & Lourido, S . SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle. Elife 13, (2024).","DOI":"10.7554\/eLife.93877.3"},{"key":"2025071600050591000_2025.07.14.664749v1.83","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2017.131"},{"key":"2025071600050591000_2025.07.14.664749v1.84","doi-asserted-by":"crossref","unstructured":"Wright, M. N. & Ziegler, A . Ranger: A fast implementation of random forests for high dimensional data in C++ and R. J. Stat. Softw. 77, (2017).","DOI":"10.18637\/jss.v077.i01"},{"key":"2025071600050591000_2025.07.14.664749v1.85","unstructured":"https:\/\/CRAN.R-project.org\/package=fastshap."},{"key":"2025071600050591000_2025.07.14.664749v1.86","doi-asserted-by":"crossref","unstructured":"Scrucca, L . On Some Extensions to GA Package: Hybrid Optimisation, Parallelisation and Islands EvolutionOn some extensions to GA package: hybrid optimisation, parallelisation and islands evolution. R J. 9, 187 (2017).","DOI":"10.32614\/RJ-2017-008"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.07.14.664749","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:06:52Z","timestamp":1768468012000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.07.14.664749"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,14]]},"references-count":86,"URL":"https:\/\/doi.org\/10.1101\/2025.07.14.664749","relation":{},"subject":[],"published":{"date-parts":[[2025,7,14]]},"subtype":"preprint"}}