https://phaidra.vetmeduni.ac.at/o:4906 Determining the effects of pseudouridine incorporation on human tRNAs Biela, Anna Anna Biela Nowak, Jakub Jakub Nowak Biela, Artur Artur Biela Mukherjee, Sunandan Sunandan Mukherjee Moafinejad, Seyed Naeim Seyed Naeim Moafinejad Maiti, Satyabrata Satyabrata Maiti Chramiec-Glabik, Andrzej Andrzej Chramiec-Glabik Mehta, Rahul Rahul Mehta Jezowski, Jakub Jakub Jezowski Dobosz, Dominika Dominika Dobosz Dahate, Priyanka Priyanka Dahate Arluison, Veronique Veronique Arluison Wien, Frank Frank Wien Indyka, Paulina Paulina Indyka Rawski, Michal Michal Rawski Bujnicki, Janusz M. Janusz M. Bujnicki Lin, Ting-Yu Ting-Yu Lin Glatt, Sebastian Sebastian Glatt 0000-0003-2815-7133 University of Veterinary Medicine Vienna Wiley journal article open access eng Transfer RNAs (tRNAs) are ubiquitous non-coding RNA molecules required to translate mRNA-encoded sequence information into nascent polypeptide chains. Their relatively small size and heterogenous patterns of their RNA modifications have impeded the systematic structural characterization of individual tRNAs. Here, we use single-particle cryo-EM to determine the structures of four human tRNAs before and after incorporation of pseudouridines (Ψ). Following post-transcriptional modifications by distinct combinations of human pseudouridine synthases, we find that tRNAs become stabilized and undergo specific local structural changes. We establish interactions between the D- and T-arms as the key linchpin in the tertiary structure of tRNAs. Our structures of human tRNAs highlight the vast potential of cryo-EM combined with biophysical measurements and computational simulations for structure-function analyses of tRNAs and other small, folded RNA domains. tRNA Cryo-EM RNA Modifications RNA Folding Molecular Dynamics Simulation http://creativecommons.org/licenses/by/4.0/ https://phaidra.vetmeduni.ac.at/api/object/o:4906/download 10.1038/s44318-025-00443-y https://phaidra.vetmeduni.ac.at/o:605 application/pdf EMBO Journal issn:1460-2075 VoR EMBO Journal 44 13 10.20 MB 2025