The tryptophan-binding pockets of Arabidopsis AGO1 facilitate amplified RNA interference via SGS3

Lopez-Marquez, D.; Arribas-Hernandez, L.; Poulsen, C.; Oksbjerg, E. D.; Bouteiller, N.; Meier, M.; Blanke, J.; del Espino, A.; Vigh, M. L.; Bressendorff, S.; Carbonell, A.; Daucke, R.; Schoof, E. M.; Vaucheret, H.; Brodersen, P.

doi:10.1101/2025.10.18.683247

bioRxiv · 10.1101/2025.10.18.683247

The tryptophan-binding pockets of Arabidopsis AGO1 facilitate amplified RNA interference via SGS3

Lopez-Marquez, D.·Arribas-Hernandez, L.·Poulsen, C.·Oksbjerg, E. D.·Bouteiller, N.·Meier, M.·Blanke, J.·del Espino, A.·Vigh, M. L.·Bressendorff, S.·Carbonell, A.·Daucke, R.·Schoof, E. M.·Vaucheret, H.·Brodersen, P.

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Abstract

ARGONAUTE (AGO) proteins associate with small RNAs to form RNA-induced silencing complexes (RISCs). Arabidopsis AGO1 effects post-transcriptional silencing by microRNAs (miRNAs) and small interfering RNAs (siRNAs) and is necessary for siRNA amplification through conversion of RISC target RNAs into double-stranded RNA by the RNA-dependent RNA Polymerase RDR6 and its mandatory cofactors SGS3 and SDE5. Many AGO proteins harbor hydrophobic pockets that interact with tryptophan residues, often surrounded by glycine (GW/WG), in intrinsically disordered regions (IDRs) of RISC cofactors. Here, we show that GW/WG dipeptides in the IDR of SGS3 and the hydrophobic pockets in AGO1 are required for fully functional RDR6-dependent siRNA amplification. We also show that this mechanism requires AGO1-specific structural elements, including positively charged residues surrounding the binding pockets, and a conserved, negatively charged patch in the IDR of SGS3. Thus, the same, conserved protein-protein interaction site is used for different purposes in distinct eukaryotic AGO proteins: the GW/WG-mediated TNRC6-Ago2 interaction is crucial for miRNA-guided silencing in metazoans whereas the GW/WG-mediated SGS3-AGO1 interaction facilitates siRNA amplification via RDR6 in plants.

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Lopez-Marquez, D., Arribas-Hernandez, L., Poulsen, C., Oksbjerg, E. D., Bouteiller, N., Meier, M., Blanke, J., del Espino, A., Vigh, M. L., Bressendorff, S., Carbonell, A., Daucke, R., Schoof, E. M., Vaucheret, H., Brodersen, P.. 2025-10-18. The tryptophan-binding pockets of Arabidopsis AGO1 facilitate amplified RNA interference via SGS3. https://doi.org/10.1101/2025.10.18.683247

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