Oksbjerg, E. D. | bioRxiv Science

bioRxiv2025

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

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.

molecular biology↗

bioRxiv2022

Distinction between small RNA-bound and free ARGONAUTE via an N-terminal protein-protein interaction site

ARGONAUTE (AGO) proteins bind to small non-coding RNAs to form RNA Induced Silencing Complexes (RISCs). In the RNA-bound state, AGO proteins are stable while RNA-free AGOs turn over rapidly. Molecular determinants unique to RNA-free AGO that allow its specific recognition and degradation remain unknown. Here, we show that a confined, linear region in Arabidopsis AGO1, the N-coil, is accessible to antibodies preferentially in the RNA-free state of AGO1. Reanalysis of hydrogen-deuterium exchange data on human Ago2 indicates similar structural flexibility of the N-coil depending on small RNA binding. Unloaded Arabidopsis AGO1 interacts with the autophagy cargo receptor ATI1 via direct contact to specific amino acid residues in the N-coil, and mutation of residues required for ATI1 interaction reduces the degradation rate of unloaded AGO1 in vivo. These results provide insight into the molecular basis for specific recognition and degradation of the RNA-free state of eukaryotic AGO proteins.

molecular biology↗