Fig. 6

SDN1 binds the AGO1 PAZ domain and triggers the 3′ end trimming of AGO1-bound miRNAs. a In vitro pull-down assays to determine the interaction between SDN1 and AGO1. Recombinant SDN1 was first purified as His-SUMO-SDN1 followed by the removal of the His-SUMO tag. SDN1 was incubated with recombinant MBP-tagged Arabidopsis AGO1/10 PAZ or MID+PIWI on beads. Western blotting using anti-SDN1 antibodies was performed to detect SDN1 retained on beads. b In vitro pull-down assays as in a except that RNase A treatment was included to determine whether the SDN1-AGO PAZ interaction is RNA-dependent. c A model of SDN1 trimming AGO1-bound miRNAs in the presence of a target RNA. The miRNA 3′ end is seized by the SDN1 DEDDh catalytic pocket with the C-terminal RRM domain binding the target strand; and SDN1 interacts with the AGO1 PAZ domain to recruit SDN1 to AGO1 RISC or to facilitate the trimming process. d–g In vitro enzymatic assays on AGO1-bound miRNAs, followed by sRNA-seq to examine the 3′ end trimming of miRNAs. d Scatter plots showing the ratio of the 3′ end truncated species (TR) to the full-length species (FL) for each miRNA detected. SDN1 caused higher levels of miRNA 3′ truncation than SDN1^D283A or SDN1^2FA. e, f Plots showing the ratio of TR species to FL species for various miRNAs. The diagonal lines represent equal levels of 3′ truncated species in the SDN1^D283A and SDN1 reactions (e), or in the SDN1^D283A and SDN1^2FA reactions (f). The miRNAs marked in blue underwent SDN1-mediated 3′ truncation (f). g Diagrams showing the status of 3′ end truncation and tailing of miR166a-3p in two biological replicates (br1 and br2). The y-axis represents the number of nucleotides truncated from the 3′ end. The x-axis represents the number of nucleotides added to the 3′ end. The size of the circles indicates the relative proportions of the species. The truncated forms of miR166a-3p generated by wild-type SDN1 are highlighted in red dashed lines