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Simultaneous nanopore profiling of mRNA m6A and pseudouridine reveals translation coordination

Nature Biotechnology (2024)Cite this article

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Abstract

N6-methyladenosine (m6A) and pseudouridine (Ψ) are the two most abundant modifications in mammalian messenger RNA, but the coordination of their biological functions remains poorly understood. We develop a machine learning-based nanopore direct RNA sequencing method (NanoSPA) that simultaneously analyzes m6A and Ψ in the human transcriptome. Applying NanoSPA to polysome profiling, we reveal opposing transcriptomic co-occurrence of m6A and Ψ and synergistic, hierarchical effects of m6A and Ψ on the polysome.

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Fig. 1: Simultaneous analysis of mRNA m6A and Ψ by NanoSPA.
Fig. 2: m6A and Ψ in translation.

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Data availability

The nanopore sequencing data of this study have been submitted to NCBI GEO (accession no. GSE230936)57. The published nanopore WT samples (replicates 1 and 2) used to validate the m6A model are GSM5467024 and GSM5467025 under GSE180656. The published WT GLORI (GSM6432590, GSM6432591) and eTAM-seq data (GSE211303, GSE211303_hela.polya.wt.ftom.ftop.rep1.deep.hits.txt.gz) are derived from the GEO database. The mESC nanopore (GSM5841801) and miCLIP2 samples (GSE163500) are derived from the GEO database. Human genome (GRCh38.p13) and annotation (gencode.v41.annotation.gff3) and mouse genome (GRCm38.primary_assembly.genome.fa) are derived from the GENCODE database (https://www.gencodegenes.org). GO information is derived from Gene Ontology Resource (http://geneontology.org).

Code availability

The NanoSPA package and tutorials are available on GitHub (https://github.com/sihaohuanguc/NanoSPA)58.

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Acknowledgements

We thank L. Zhang and C. He for providing analysis of m6A-SAC-seq data before publication and D. Pan for contribution to coding. This work was supported by NIH (no. RM1 HG008935 to T.P.).

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Author notes

  1. These authors contributed equally: Sihao Huang, Adam C. Wylder.

Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, IL, USA

    Sihao Huang & Tao Pan

  2. Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL, USA

    Adam C. Wylder

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Contributions

S.H. performed all computational work including NanoSPA pipeline development and sequencing data analysis. A.C.W. performed all experimental work including siRNA knockdown, polysome profiling and nanopore sequencing. S.H. and T.P. conceived the project. S.H., A.C.W. and T.P. designed the experiments and wrote the paper.

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Correspondence to Tao Pan.

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Huang, S., Wylder, A.C. & Pan, T. Simultaneous nanopore profiling of mRNA m6A and pseudouridine reveals translation coordination. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02135-0

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