3' Nucleotide Asymmetry Directs miRNA Strand Selection

Medley, J. C.; Kurosu Moriya, S.; Ouyang, H.; Crawshaw, H.; Zhang, S. Y.; Panzade, G.; Sydzyik, W. J.; Sydzyik, J. T.; Bhandari, M.; Hammell, C. M.; Zinovyeva, A.

doi:10.1101/2025.10.15.682714

bioRxiv · 10.1101/2025.10.15.682714

3' Nucleotide Asymmetry Directs miRNA Strand Selection

Medley, J. C.·Kurosu Moriya, S.·Ouyang, H.·Crawshaw, H.·Zhang, S. Y.·Panzade, G.·Sydzyik, W. J.·Sydzyik, J. T.·Bhandari, M.·Hammell, C. M.·Zinovyeva, A.

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Abstract

Accurate microRNA (miRNA) strand selection is essential for defining the regulatory landscape of the miRNA-induced silencing complex (miRISC). While 5' nucleotide identity and duplex thermodynamics have been proposed to bias strand choice, these features cannot fully explain in vivo strand preferences. Here, we uncover a conserved and previously unrecognized role for 3' nucleotide asymmetry in directing miRNA strand selection in Caenorhabditis elegans and human cells. A favorable 3' terminal nucleotide on the passenger strand promotes selective loading of the opposing guide strand into miRISC, revealing a cooperative interplay between 5' and 3' terminal asymmetries that ensures precise strand discrimination. These findings establish a unified, evolutionarily conserved mechanism for miRNA duplex sorting and expand the fundamental rules governing small RNA biogenesis.

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Medley, J. C., Kurosu Moriya, S., Ouyang, H., Crawshaw, H., Zhang, S. Y., Panzade, G., Sydzyik, W. J., Sydzyik, J. T., Bhandari, M., Hammell, C. M., Zinovyeva, A.. 2025-10-16. 3' Nucleotide Asymmetry Directs miRNA Strand Selection. https://doi.org/10.1101/2025.10.15.682714

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