Disturbed pseudouridine metabolism is linked to human genetic diseases. Pseudouridine synthase 3 (PUS3) is responsible for the formation of pseudouridine at position 38/39 in tRNAs. The Leidel and Glatt labs determined four Cryo-EM structures of the apo form of human PUS3 and bound to three tRNAs, allowing them to describe its substrate selection mechanism which is different from other PUS enzymes. Through biochemical assays using mutations based on the structure and derived from patients they could confirm the conclusions drawn from the structure regarding the enzyme's mechanism. Also, they could that PUS3 in cells does not seem to modify mRNAs. Their findings allow them to explain how mutations in it lead to human disease. The paper "The molecular basis of tRNA selectivity by human pseudouridine synthase 3" has been published in Molecular Cell.

Highlights

• Single-particle cryo-EM structures reveal how human PUS3 recognizes tRNAs
• Two distinct interfaces in PUS3 are key for tRNA binding and positioning
• PUS1- but not PUS3-dependent pseudouridine sites are found in RNAs other than tRNAs
• Patient-derived mutations link PUS3’s tRNA modification activity to human disease

Summary
Pseudouridine, 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 pseudouridine 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.

Read the Publication in Molecular Cell (Open Access)

Website Leidel Lab

Figure, highlights and summary from Lin, Kleemann et al. (2024) Mol Cell published under a CC BY-NC 4.0 license.