The molecular basis of tRNA selectivity by human pseudouridine synthase 3

Lin, Ting-Yu; Kleemann, Leon; Jezowski, Jakub; Dobosz, Dominika; Rawski, Michal; Indyka, Paulina; Wazny, Grzegorz; Mehta, Rahul; Chramiec-Glabik, Andrzej; Koziej, Lukasz; Ranff, Tristan; Fufezan, Christian; Wawro, Mateusz; Kochan, Jakub; Bereta, Joanna; A. Leidel, Sebastian; Glatt, Sebastian

Abstract

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Citation

Lin, T.-Y., Kleemann, L., Jezowski, J., Dobosz, D., Rawski, M., Indyka, P., Wazny, G., Mehta, R., Chramiec-Glabik, A., Koziej, L., Ranff, T., Fufezan, C., Wawro, M., Kochan, J., Bereta, J., A. Leidel, S., & Glatt, S. (2024). The molecular basis of tRNA selectivity by human pseudouridine synthase 3. Molecular Cell, 84(13), 2472-2489.e8. https://doi.org/10.1016/j.molcel.2024.06.013

• Journal Article Type: Article
• Acceptance Date: Jun 13, 2024
• Online Publication Date: Jul 11, 2024
• Publication Date: Jul 11, 2024
• Deposit Date: Jul 23, 2024
• Journal: Molecular Cell
• Print ISSN: 1097-2765
• Electronic ISSN: 1097-4164
• Publisher: Cell Press
• Peer Reviewed: Peer Reviewed
• Volume: 84
• Issue: 13
• Pages: 2472-2489.e8
• DOI: https://doi.org/10.1016/j.molcel.2024.06.013
• Keywords: Intramolecular Transferases, Pseudo-seq, PUS3, HEK293 Cells, anticodon stem loop, cryo-EM structure, Protein Binding, PUS1, Hydro-Lyases - metabolism - genetics - chemistry, RNA, Messenger - genetics - metabolism, Intellectual Disability - genetics - metabolism - enzymology, tRNA modification, Catalytic Domain, Pseudouridine - metabolism - genetics, Models, Molecular, transcriptome, Cryoelectron Microscopy, RNA, Transfer - metabolism - genetics, Substrate Specificity, Mutation, Humans, pseudouridine synthase
• Public URL: https://durham-repository.worktribe.com/output/2610522
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S1097276524005203?via%3Dihub