Fig. 5.

Depletion of endogenous NtSGS3 in BYL destabilizes cleavage fragment–miR173–RISC complexes. (A) Copurification of TAS2-derived RNAs with FLAG-NtAGO1 after depletion of endogenous NtSGS3. The BYL-based reaction mixtures containing FLAG-NtAGO1 and 50 nM miR173/miR173* were incubated with affinity-purified anti-GUS or anti-NtSGS3 antibodies (or mock-conjugated), treated with protein A Sepharose, and then mixed with 5 nM ³²P-labeled target RNAs. From these mixtures, FLAG-NtAGO1 was immunoprecipitated with anti-FLAG antibody. RNA was extracted from the immunoprecipitates and analyzed by denaturing 5% PAGE and autoradiography. RNA from input samples was analyzed in parallel (Upper two panels). Endogenous NtSGS3 or FLAG-NtAGO1 in each reaction mixture was detected with anti-NtSGS3 or anti-FLAG antibodies, respectively (Lower two panels). (B) In vitro complementation of endogenous NtSGS3 depletion by AtSGS3-myc with regard to stabilization of RISC-cleaved fragments and cleaved RNA–RISC complexes. BYL-based reaction mixtures containing miR171- or miR173-bound FLAG-NtAGO1 were incubated with anti-NtSGS3 antibodies, treated with protein A Sepharose, and then combined with mock-translated and mock-depleted lysate, mock-translated and NtSGS3-depleted lysate, or AtSGS3-myc mRNA-translated and NtSGS3-depleted lysate. ³²P-labeled target RNAs (5 nM) were added to each reaction mixture, and FLAG-NtAGO1 was immunoprecipitated with anti-FLAG antibody. RNA was extracted from the immunoprecipitates and analyzed by denaturing 5% PAGE and autoradiography. RNA from input samples was analyzed in parallel (Upper). Endogenous NtSGS3, FLAG-NtAGO1, and AtSGS3-myc in the mixtures were detected with anti-NtSGS3, anti-FLAG, and anti-myc antibodies, respectively (Lower).