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Direct profiling of non-adenosines in poly(A) tails of endogenous and therapeutic mRNAs with Ninetails

Author

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  • Natalia Gumińska

    (International Institute of Molecular and Cell Biology)

  • Katarzyna Matylla-Kulińska

    (International Institute of Molecular and Cell Biology
    University of Warsaw)

  • Paweł S. Krawczyk

    (International Institute of Molecular and Cell Biology)

  • Michał Maj

    (NatWest Group)

  • Wiktoria Orzeł

    (International Institute of Molecular and Cell Biology
    University of Warsaw)

  • Zuzanna Mackiewicz

    (International Institute of Molecular and Cell Biology)

  • Aleksandra Brouze

    (International Institute of Molecular and Cell Biology)

  • Seweryn Mroczek

    (International Institute of Molecular and Cell Biology
    University of Warsaw)

  • Andrzej Dziembowski

    (International Institute of Molecular and Cell Biology
    University of Warsaw)

Abstract

Stability and translation of mRNAs, both endogenous and therapeutic, is determined by poly(A) tail. Direct RNA sequencing enables single-molecule measurements of poly(A) lengths, avoiding amplification bias. It also holds potential for observation of non-adenosines within poly(A), known to influence mRNA fate. However, there is no computational method to detect composite tails in Direct Sequencing data. To address this gap, we introduce the Ninetails, a neural network-based tool that accurately identifies and quantifies non-adenosines in poly(A) tails. Examination of different biological contexts revealed widespread non-adenosine decorations, with frequencies influenced by the origin of poly(A) tails differing by mRNA class, cell type, and species. Notably, substrates of cytoplasmic TENT5-polymerases and mitochondrially encoded mRNAs are enriched in composite tails. For mRNA therapeutics, we show that the composition of poly(A) tails in mRNA vaccines is dynamic during its cellular lifetime and that the manufacturing protocol of synthetic mRNAs affects the purity of poly(A) tails.

Suggested Citation

  • Natalia Gumińska & Katarzyna Matylla-Kulińska & Paweł S. Krawczyk & Michał Maj & Wiktoria Orzeł & Zuzanna Mackiewicz & Aleksandra Brouze & Seweryn Mroczek & Andrzej Dziembowski, 2025. "Direct profiling of non-adenosines in poly(A) tails of endogenous and therapeutic mRNAs with Ninetails," Nature Communications, Nature, vol. 16(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57787-6
    DOI: 10.1038/s41467-025-57787-6
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