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Differential roles of human PUS10 in miRNA processing and tRNA pseudouridylation

Abstract

Pseudouridine synthases (PUSs) are responsible for installation of pseudouridine (Ψ) modification in RNA. However, the activity and function of the PUS enzymes remain largely unexplored. Here we focus on human PUS10 and find that it co-expresses with the microprocessor (DROSHA–DGCR8 complex). Depletion of PUS10 results in a marked reduction of the expression level of a large number of mature miRNAs and concomitant accumulation of unprocessed primary microRNAs (pri-miRNAs) in multiple human cells. Mechanistically, PUS10 directly binds to pri-miRNAs and interacts with the microprocessor to promote miRNA biogenesis. Unexpectedly, this process is independent of the catalytic activity of PUS10. Additionally, we develop a sequencing method to profile Ψ in the tRNAome and report PUS10-dependent Ψ sites in tRNA. Collectively, our findings reveal differential functions of PUS10 in nuclear miRNA processing and in cytoplasmic tRNA pseudouridylation.

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Fig. 1: PUS10 modulates the expression levels of miRNAs.
Fig. 2: PUS10 regulates the expression of miRNAs by promoting pri-miRNA processing.
Fig. 3: PUS10 promotes miRNA processing in a catalytically independent manner.
Fig. 4: PUS10 binds to pri-miRNAs and interacts with microprocessor.
Fig. 5: Identification of PUS10-dependent Ψ sites in tRNA.
Fig. 6: The tRNA pseudouridylation activity of PUS10 is restrained in the cytoplasm.

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Data availability

The sequencing data obtained in this study have been deposited in the NCBI Gene Expression Omnibus under accession number GSE124558.

Code availability

All custom code and scripts are available from the authors upon request.

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Acknowledgements

We thank Y. Wang, F. Duan, X. Zhang and X. Lv (Peking University) for sharing antibodies, plasmids and cell lines. We are indebted to Y. Wang, Y. Liu, P. Du for advice on miRNA. We also thank X. Li and K. Wang (Peking University) for sharing purified recombinant wild-type AlkB and AlkB D135S mutant proteins; K. Yin for the suggestion on cell-growth assays; and X. Xiong for the suggestion on bioinformatic analysis. We thank the National Center for Protein Sciences at Peking University for assistance with RT–qPCR and flow cytometry. Part of the analysis was performed on the Computing Platform of the Center for Life Science. This work was supported by the National Natural Science Foundation of China (grants 21825701, 91740112 and 31861143026 to C.Y. and grant 81622035 to M.L.), the Joint Laboratory of International Scientific and Technological Cooperation and China Postdoctoral Science Foundation (grant 2018M641076 to J.S.).

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Authors and Affiliations

Authors

Contributions

C.Y. and J.S. conceived the project and designed the experiments. J.S. performed microarray experiments, validation of the expression of miRNA and pri-miRNA by RT–qPCR and northern blot, rescue assays, rRNA(−) nuclear RNA-seq, polyA(+) RNA-seq and PAR-CLIP. B.L. and J.S. generated the PUS10 KO cell line. Y.Z. and B.X. performed cell-differentiation experiments. J.S. and Y.Z. performed in vitro processing assays, native-gel-shift assays, activity assays, cell-proliferation assays and ribosome profiling. J.S. designed DM–Ψ-seq, and J.S. and Y.Z. performed DM–Ψ-seq. H.M. and J.S. performed co-expression analysis and microarray analysis. C.Z. and H.M. analyzed PAR-CLIP results and C.Z. analyzed RNA-seq and ribosome profiling data. J.S. and C.Z. analyzed DM–Ψ-seq data. C.Y. and M.L. supervised the project. C.Y., J.S., Y.Z. and C.Z. wrote the manuscript with contributions from all authors. J.P. discussed and commented on the manuscript.

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Correspondence to Mo Li or Chengqi Yi.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–17

Reporting Summary

Dataset 1

Significantly downregulated miRNA upon PUS10 knockdown as determined by microarray.

Dataset 2

The binding clusters identified by PUS10 PAR-CLIP.

Dataset 3

Ψ sites identified by DM–Ψ-seq

Dataset 4

RT–qPCR primers, probes in northern blotting, and RT-primers of CMC primer-extension assay.

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Song, J., Zhuang, Y., Zhu, C. et al. Differential roles of human PUS10 in miRNA processing and tRNA pseudouridylation. Nat Chem Biol 16, 160–169 (2020). https://doi.org/10.1038/s41589-019-0420-5

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