Abstract
Ribosome biogenesis requires the synthesis and sequential processing of precursor rRNAs (pre-rRNAs) into mature rRNAs. Traditional methods such as northern blotting and metabolic labeling provide limited resolution. Here, we present NanoRibolyzer, a nanopore-based long-read sequencing approach that enables ab initio identification and quantification of rRNA precursors while simultaneously mapping RNA modifications. Using supervised and unsupervised mapping, we detect both known and previously uncharacterized pre-rRNAs and delineate cleavage events at single-nucleotide resolution. A simple cell-fractionation protocol further separates nuclear and cytoplasmic pre-rRNAs, allowing spatial deconvolution of processing pathways. By projecting each sequenced molecule in a two-dimensional space using its starting and ending coordinates, we generate an intuitive representation in which the activity of the 5′ → 3′ and 3′ → 5′ exoRNases can be tracked as they mature pre-rRNAs one nucleotide at a time. Targeted knockdowns of ribosome-assembly factors quantify accumulation of intermediates and reveal condition-specific processing “fingerprints” with biomarker potential. High-resolution re-analysis of known factors uncovers unexpected functions. Additionally, pseudouridine mapping shows that the primary 47S transcript is extensively modified, whereas aberrant intermediates (34S and 36S-C) are hypomodified. With its high resolution and unique discovery mode, NanoRibolyzer provides new insights into rRNA processing and modification, greatly advancing our understanding of ribosome biogenesis.
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Data availability
Nanopore sequencing data were deposited in the European Nucleotide Archive (ENA) under project accession number PRJEB82698 [ebi.ac.uk/ena/browser/view/PRJEB82698] and are publicly available as of the date of publication. Owing to repository file size limitations, samples ERS24812595 and ERS24812596 were randomly subsampled before public deposition. If the entire dataset of those samples is required, please contact the authors. All data, including raw numbers for graphs, are available in the Source Data file or in the Supplementary Information. Source data are provided with this paper. Source data are provided with this paper.
Code availability
All scripts and code used in this work have been made available on GitHub (https://github.com/stegiopast/wf-nanoribolyzer). The preprocessing pipeline for single samples is part of the NextFlow pipeline. Code for downstream analysis is stored in Jupyter Notebooks in the GitHub repository.
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Acknowledgements
This work was partly funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation); project no. 439669440 TRR319 RMaP, TP A05/C01/C03 to M.H. and S.M, TP B07 to T.B., TP A07 to S.G. and L.L., and TP C04 to S.G and S.P., T.B. and S.G. acknowledge funding from the ReALity initiative of the Johannes Gutenberg University Mainz. Research in the Lab of D.L.J.L. was supported by the Belgian Fonds de la Recherche Scientifique (F.R.S./FNRS), EOS [CD-INFLADIS, grant n°40007512], Région Wallonne (SPW EER) Win4SpinOff [RIBOGENESIS], the COST actions EPITRAN (CA16120) and TRANSLACORE (CA21154), the European Joint Program on Rare Diseases (EJP-RD) RiboEurope and DBAGeneCure. S.G. and L.L acknowledge funding by the Boehringer Ingelheim Stiftung. S.G. acknowledges funding by the M3odel initiative from the Forschungsinitiative Rheinland-Pfalz.
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S.P. developed and implemented the NanoRibolyzer pipeline; S.P. and L.L. analyzed the data. T.B., L.W., and S.P. performed nuclei isolation and RNA isolation experiments. L.W. performed the knockdown experiments and northern blots. S.M. performed the in vitro transcription of 18S and 28S rRNA. T.B. and S.P. prepared and sequenced the nanopore-seq libraries. T.B. and D.L. conceived and supervised the work, with the assistance of S.G., M.H., and B.L., who provided valuable input and feedback in various discussions. T.B., D.L., and S.P. wrote the paper, with contributions from all the authors.
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M.H. serves as a consultant for Moderna, Inc. All other authors declare no competing interests.
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Pastore, S., Wacheul, L., Lehmann, L. et al. Mapping human pre-rRNA processing and modification at single nucleotide resolution using long read nanopore sequencing. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71164-x
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DOI: https://doi.org/10.1038/s41467-026-71164-x
