Abstract
Pseudouridine (Ψ) is an abundant modification in small RNA catalyzed by multiple pseudouridine synthases (PUSs). However, the substrate specificity of human PUSs remains elusive. Here, we adopted PRAISE, a quantitative Ψ detection method, to profile pseudouridylation in small RNA, including cytosolic and mitochondrial tRNAs, snRNA, and snoRNA. We found that snoRNA pseudouridylation is mediated not only by RNA-guided DKC1, but also by the stand-alone enzyme PUS7 at a specific site. Interestingly, several PUS enzymes, including PUS1, RPUSD1, and PUS7, which install nearby Ψ sites within tRNA anticodon stem-loop, can influence pseudouridylation catalyzed by other PUSs, revealing an unrecognized interplay during Ψ formation. For the three RluA family enzymes, RPUSD1 catalyzes the canonical Ψ30 in tRNA-Ile and Ψ72 in tRNA-Arg isoacceptors. RPUSD2 pseudouridylates Ψ31 of mt-tRNALeu(CUN), Ψ32 of mt-tRNAPro and mt-tRNACys, whereas RPUSD3 lacks tRNA activity. Together, our quantitative Ψ profiling characterized PUS tRNA substrates and revealed unexpected PUS interplay.
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Data availability
The sequencing data supporting the results of this study are available in public repositories. The main raw and processed sequencing data have been deposited into the NCBI Gene Expression Omnibus (GEO) under the accession number GSE299274. Additional sequencing data have been deposited in the Genome Sequence Archive (GSA) at the CNCB-NGDC under PRJCA050602. Source data are provided with this paper.
Code availability
The analysis scripts in this study are publicly available in the GitHub repository at https://github.com/LiuWenQing657/PsiUInterplayAnalysis, which is also cited here71.
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Acknowledgements
The authors would like to thank Dr. Xiaoting Zhang, Dr. Hanxiao Sun, Dr. Jinmin Yang, and Jinghan Zhou for discussions, and Dr. Hongxia Chen for providing the PUS7L knockout cell line. This work was supported by the National Key R&D Program of China (2021YFC2302400 to M.Z. and 2023YFC3402200 to C.Y.), the National Natural Science Foundation of China (22425071 to C.Y. and 61575008 to M.Z.), the Beijing National Science Foundation of China (Z231100002723005 to C.Y.), and the Natural Science Foundation of Sichuan Province of China (2026NSFSC0869 to B.H.). This work was supported by the New Cornerstone Science Foundation through the XPLORER PRIZE. This work was supported by the Open Research Fund of the National Center for Protein Sciences at Peking University and the Chinese Universities Scientific Fund 2025TC012.
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C.Y. and M.Z. conceived the project and designed the experiments; M.Z., C.Y., and W.L. wrote the manuscript; M.Z. performed the experiments with the help of L.W. and Y.D.; W.L. and Y.M. designed and performed the bioinformatics analysis for NGS samples with the help of B.H. B.L. constructed the PUS1L and three RPUSD knockout cell lines. Y.Z. performed the immunofluorescence assays.
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Liu, W., Ma, Y., Wang, L. et al. Quantitative analysis of small RNA pseudouridylation reveals interplay of PUS enzymes in tRNA anticodon stem-loop. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69177-7
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DOI: https://doi.org/10.1038/s41467-026-69177-7
