The molecular basis of tRNA selectivity by human pseudouridine synthase 3

Summary

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.

Graphical abstract

Highlights

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  Single-particle cryo-EM structures reveal how human PUS3 recognizes tRNAs

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  Two distinct interfaces in PUS3 are key for tRNA binding and positioning

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  PUS1- but not PUS3-dependent Ψ sites are found in RNAs other than tRNAs

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  Patient-derived mutations link PUS3’s tRNA modification activity to human disease

Abstract

Lin, Kleemann et al. provide a comprehensive structure-function analysis of human PUS3, which catalyzes the conversion of uridine to pseudouridine (Ψ). PUS3 forms a homodimer to selectively bind and specifically modify tRNAs. No PUS3-dependent Ψs were detected in mRNA, linking the associated human diseases to tRNAs.