Abstract
RNA modifications play a crucial role in regulating cellular functions. Among the most abundant modifications in the human transcriptome are pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C). However, the interplay between these modifications remains poorly understood due to limited integrative studies. To address the gap, we utilized nanopore direct RNA sequencing to quantify the stoichiometry of Ψ, m6A, and m5C after depleting the pseudouridine synthases PUS7 or DKC1. We used the custom tool NanoPsiPy to quantify pseudouridine by analyzing differential U-to-C base-calling errors in nanopore sequencing data. For m6A and m5C, we applied the established tool CHEUI to conduct stoichiometry differential analysis. Our investigation identified both known and novel pseudouridylation sites in tRNA, rRNA, and mRNA targeted by PUS7 or DKC1. Integrative analysis revealed that depletion of PUS7 or DKC1 reduced pseudouridylation levels while simultaneously increasing global m6A and m5C levels, with functional implications for mRNA translation regulation. These findings suggest that pseudouridylation may play an active role in repressing m6A and m5C modifications. This study demonstrates the analytical power of nanopore direct RNA sequencing for investigating co-regulation of RNA modifications.
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Data availability
The BioProject accession number for nanopore RNA-seq data reported in this paper is PRJNA1220613. All other raw data are available upon request from the corresponding authors.
Code availability
The NanoPsiPy package and tutorials are available on GitHub (https://github.com/vetmohit89/NanoPsiPy).
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Acknowledgements
A.K. was partly supported by a Concept Award (Award HT9425-23-1-0339) and an Early Career Award (HT9425-23-1-0783) from the Department of Defense. We would like to thank Shane Rich-New and IT Research Computing Team at the University of Alabama at Birmingham for NGS support. This work was supported by the National Institutes of Health under grant R01CA190429 to H.-F.D. and by NIH grants R00CA259526 to Z.S. and T32GM135028 to K.M.
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M.B. and H.-F.D. conceived the study and designed the experiments with contributions from A.G., J.D., A.K., A.G., Z.S., K.M., and S.S. M.B. and K.M. performed nanopore RNA sequencing and data analysis with assistance from A. Gibson. A.K. performed PUS7 purification. A.G. and J.D. performed cell culture and immunoblotting. M.B. and H.-F.D. wrote the paper with contributions from S.S. and Z.S. H.-F.D. supervised and provided funding for the project. All authors read the manuscript and approved its contents.
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Bansal, M., Gupta, A., Ding, J. et al. Integrative analysis of nanopore direct RNA sequencing data reveals a global impact of pseudouridylation on m6A and m5C modifications. npj Precis. Onc. (2026). https://doi.org/10.1038/s41698-026-01278-4
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DOI: https://doi.org/10.1038/s41698-026-01278-4
