The N-coil and the globular N-terminal domain of plant ARGONAUTE1 are interaction hubs for regulatory factors

Abstract

The effector complex of RNA interference (RNAi) contains at its core an ARGONAUTE (AGO) protein bound to a small guide RNA. AGO proteins adopt a two-lobed structure in which the N-terminal (N) and Piwi-Argonaute-Zwille (PAZ) domains make up one lobe, while the middle (MID) and Piwi domains make up the other. Specific biochemical functions of PAZ, MID and Piwi domains of eukaryotic AGO proteins have been described, but the functions of the N-terminal domain remain less clear. Here, we use yeast two-hybrid screening with the N-terminal domain of the founding member of the AGO protein family, arabidopsis AGO1, to reveal that it interacts with many factors involved in regulated proteolysis. Interaction with a large group of proteins, including the autophagy cargo receptors ATI1 and ATI2, requires residues in a short, linear region, the N-coil, that joins the MID-Piwi lobe in the three-dimensional structure of AGO. In contrast, the F-box protein AUF1 interacts with AGO1 independently of the N-coil and requires distinct residues in the globular N domain itself. Mutation of AGO1 residues necessary for interaction with protein degradation factors in yeast stabilizes reporters fused to the AGO1 N-terminal domain in plants, supporting their in vivo relevance. Our results define distinct regions of the N domain implicated in protein-protein interaction, and point to a particular importance of the AGO1 N-coil as a site of interaction with regulatory factors.

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

“…This may indicate that the conformation of the N-coil revealed in the crystal structures of AGO-small RNA complexes is but one of several possible conformations of this region. Second, specific residues in the N-coil of Arabidopsis AGO1 (Lys178, Lys185, Gly186 and Lys190) are required for yeast two-hybrid interaction of the N-domain of AGO1 with factors implicated in regulated proteolysis, including the autophagy cargo receptor ATI1 23 . Combining the indication of alternative N-coil conformations, the known instability of unloaded AGO proteins, and the N-coil-dependent two-hybrid interactions with regulated proteolysis factors, we hypothesize that the N-coil is accessible in the unloaded conformation of AGO1, but becomes attached to the Piwi domain as part of the loading process.…”

“…To test the second prediction that regulated proteolysis factors directly bind the N-coil, we chose the interaction between a fragment of AGO1 comprising the N-coil and the globular N-domain (NcGN) and the N-terminal IDR of the autophagy cargo receptor ATI1 27 as model system. Using yeast two-hybrid assays, this interaction was shown to depend on the N-coil residues Lys178, Lys185 and Lys190 23 . We first expressed and purified the IDR of ATI1 27 and wild type and K178E mutant versions of the NcGN of AGO1 fused to His 6 -SUMO (His 6 -SUMO-NcGN AGO1 , Figure 4A), and used these purified proteins for quantitative binding assays by microscale thermophoresis.…”

“…Because heterologous expression of AGO1 failed, even with the use of a variety of expression hosts ( E. coli , baculovirus/ Sf-9, Schizosaccharomyces pombe ) and affinity/solubilization tags, we used bimolecular fluorescence complementation (BiFC) assays upon transient expression in Nicotiana benthamiana for assessment of ATI1 interaction with AGO1 WT and AGO1 Y691E . The close AGO1 paralogue, AGO10, whose NcGN part interacts much less strongly with ATI1 in the two-hybrid assay than AGO1 23 , was used as a control expected to yield substantially less specific signal than AGO1. With AGO1 WT fused to the C-terminal part of YFP and ATI1 fused to its N-terminal part (C-YFP-AGO1 WT and N-YFP-ATI1), weak, but specific signal was detected in association with the endoplasmic reticulum (ER; Figure 5A), consistent with previous reports of ATI1-localization to ER-associated ATI-bodies, and with peripheral ER-association of AGO1 29,30 .…”

“…Second, specific residues in the N-coil of Arabidopsis AGO1 (Lys178, Lys185, Gly186 and Lys190) are required for yeast two-hybrid interaction of the N-domain of AGO1 with factors implicated in regulated proteolysis, including the autophagy cargo receptor ATI1 23 .…”

“…The close AGO1 paralogue, AGO10, whose NcGN part interacts much less strongly with ATI1 in the two-hybrid assay than AGO1 23 , was used as a control expected to yield substantially less specific signal than AGO1. With AGO1 WT fused to the C-terminal part of YFP and ATI1 fused to its N-terminal part (C-YFP-AGO1 WT and N-YFP-ATI1), weak, but specific signal was detected in association with the endoplasmic reticulum (ER; Figure 5A), consistent with previous reports of ATI1-localization to ER-associated ATI-bodies, and with peripheral ER-association of AGO1 29,30 .…”