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 undergo regulated proteolysis or associate with chaperones en route to RISC maturation. Molecular determinants unique to RNA-free AGO that allow its specific recognition and degradation remain poorly characterized. Here, we show that the Arabidopsis autophagy cargo receptor ATI1 interacts with unloaded AGO1 via multiple interaction sites that include direct contact to a confined, linear region in AGO1, the N-coil. The N-coil is accessible to antibodies preferentially in the RNA-free state of AGO1, and influences its degradation rate in vivo. These results provide insight into the molecular basis for specific recognition of the RNA-free state of eukaryotic AGO proteins, and introduce a new tool for its further study.