Functional Analysis of AGO2 Catalytic Residues in Ternary Complex Stabilization and Target Transcript Slicing.

(A) Foldbacks of wild-type MIR390a and MIR173a and of artificial miRNAs  amiR173 and amiR173-5′A engineered within the MIR390a foldback. Green and black, miRNA guide and miRNA* strands, respectively. Mutagenized positions are shown in red.

(B) Immunoprecipitations with HA-AGO2 proteins. Input and immunoprecipitation fractions from N. benthamiana following coexpression of 35S:amiR173-5′A and 35S:TAS1c-A388T with 35S:GUS, 35S:HA-AGO2-DDD, 35S:HA-AGO2-DAD, 35S:HA-AGO2-DDA, and 35S:HA-AGO2-DDH, as well as coexpression of 35S:amiR173 with 35S:TAS1c-A388T and 35S:HA-AGO2-DDD (wt), were analyzed. Top, ethidium bromide–stained RT-PCR products corresponding to a noncleaved fragment from the TAS1c-A388T target transcript. Middle, amiR173/amiR173-5′A blot is shown as control for HA-AGO2 binding and immunoprecipitation selectivity. Bottom, HA-AGO2 blots. l-Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) stained membrane was included as input loading and immunoprecipitation control. The catalytic residues are shown in bold, those found in wild-type AGO2 are in black, and those modified are in red. nt, nucleotides.

(C) Ethidium bromide–stained 5′RACE products corresponding to the 3′ cleavage product of TAS1c-A388T targets cleaved by amiR173-5′A/AGO2 complexes. N. benthamiana actin RT-PCR products are shown as control.

(D) Proportion of cloned 5′RACE products corresponding to cleavage within TAS1c-A388T transcripts at the amiR173-5′A targeted site in assays with HA-AGO2 forms.