Abstract
MicroRNAs direct Argonaute proteins to repress complementary target mRNAs via mRNA degradation or translational inhibition. While mammalian miRNA targeting has been well studied, the principles by which Drosophila miRNAs bind their target RNAs remain to be fully characterized. Here, we use RNA Bind-n-Seq to systematically identify binding sites and measure their affinities for five highly expressed Drosophila miRNAs. Our results reveal a narrower range of binding site diversity in flies compared to mammals, with fly miRNAs favoring canonical seed-matched sites and exhibiting limited tolerance for imperfections within these sites. We also identified non-canonical site types, including nucleation-bulged and 3′-only sites, whose binding affinities are comparable to canonical sites. These findings establish a foundation for future computational models of Drosophila miRNA targeting, enabling predictions of regulatory outcomes in response to cellular signals, and advancing our understanding of miRNA-mediated regulation in flies.
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Data availability
The data supporting the findings of this study are available from the corresponding authors upon request. RBNS sequencing data have been deposited at National Center for Biotechnology Information Sequence Read Archive and are publicly available using accession number PRJNA1185003. The source data underlying Figs. 1c, 2b–c, and 5b, Supplementary Figs. 2b and 5c–d are provided as a Source Data file. Source data are provided with this paper.
Code availability
This study did not generate new code. To estimate KD values, we used the code previously published25 (https://figshare.com/articles/software/MicroRNA-binding_thermodynamics_and_kinetics_by_RNA_Bind-n-Seq/19180952).
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Acknowledgements
We thank members of the Zamore and Jouravleva laboratories for critical comments on the manuscript. We thank the KYOTO Drosophila Stock Center (Drosophila Genomics and Genetic Resources, Kyoto Institute of Technology) for providing fly stocks used in this study. This work was supported in part by National Institutes of Health grant R35 GM136275 to P.D.Z. and by ATIP-Avenir funding (CNRS Biology) to K.J.
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Conceptualization, J.V.B, K.J., and P.D.Z.; Methodology, J.V.B. and K.J.; Investigation, J.V.B. and K.J.; Formal analysis, K.J. and J.V.B.; Writing—original draft, K.J. and J.V.B.; Writing—review and editing, K.J., J.V.B., and P.D.Z.; Supervision, K.J. and P.D.Z.; Funding acquisition, K.J. and P.D.Z.
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Vega-Badillo, J., Zamore, P.D. & Jouravleva, K. Biochemical principles of miRNA targeting in flies. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68360-0
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DOI: https://doi.org/10.1038/s41467-026-68360-0
