Figure 3. Rearrangement of PIWI subdomains during RISC assembly.
(A) Different arrangements of PIWI-helical subdomain and MID domain. (Top) The crystal structures of the guide-bound AG04 and the apo-form NcQDE2 (PDB ID: 2YHA) are superimposed on their PIWI-catalytic subdomains (gray). The pseudo-catalytic residue of AG04, R809 (green), and the catalytic residue of NcQDE2, D890 (pink), are depicted as a stick model on α18 (see also Figure 1D). The transition from the apo-NcQDE2 to that of the guide-bound AG04 is shown by red arrows. For clarity, loops L31 and L32 and the N-PAZ lobe of AG04 are not shown. (Bottom) Schematic of the MID and PIWI domains.
(B) Interactions between the two PIWI subdomains of NcQDE2 (top), AG04 (middle), and AG02 (bottom). Residues involved in the hydrophobic interaction are shown as stick models with their van der Waals radii (dots). Colors as in (A).
(C) Seed region of guide RNA tying together two PIWI subdomains of AG04. For clarity, the N-PAZ lobe is not shown. The PIWI-catalytic subdomain is shown as a surface model. The MID domain and the PIWI-helical subdomain are depicted as ribbon models. The guide RNA is colored in red. Colors as in (A).
(D) Target cleavage assay of AG02 and mutants. The amount of AGOs used for target cleavage assays (right) was adjusted based on the western blot with anti-FLAG antibody (left).
(E) Duplex loading of AG02 and mutants. See also Figure S4E.
(F) Co-immunoprecipitation of TNRC6A with AG02 and mutants. The amount of AGOs used to detect co-immunopurified TNRC6A with anti-TNRC6A antibody (right) was adjusted based on the western blot with anti-FLAG antibody (left).
Experiments in D and F were performed at least three times. A representative gel is shown.