(A–C) Ectopic expression of catalytically-inactive RICE1 mutants (A and B) and downregulation of RICEs (C) decreased miRNA accumulation in RISC. Western blot analyses were done with the crude extract (Input) and the IP products using anti-AGO1 antibody (top panels). Actin serves as a loading control. sRNA blot analyses of sRNA recovered from AGO1 IP were conducted with 32P-labelled probe against miR166. The ratio of miR166/AGO1 in wild-type Ler was arbitrarily designated as 1.0, and the relative amount in each sample was normalized to that of control plants. (D) Quantitative PCR showed the concurrent reduction of miRNA and miRNA* abundance in the transgenic plants expressing artificial miRNAs specifically targeting RICE1, RICE2 or both. The relative level of miRNA and miRNA* was normalized to that in Ler plants where the amount was arbitrarily assigned a value of 1 with ±SD from at least three experiments, asterisks indicate statistically significant differences between wild type and transgenic plants. (E) sRNA blot analysis of highly-enriched low-molecular-weight RNAs prepared from wild-type and RICE-compromised transgenic lines. Numerous 21-nt and truncated oligo probes complementary to miR165 (a–b)*, miR166 (a–g)*, miR159b*, miR160c*, miR162b*, miR164b*, miR167b*, miR168a*, and miR171a* were pooled for sRNA blot. Note: The region marked in red box is the place where 9-nt and 12-nt miRNA* fragments would have been detected, if present. (F) In vitro RISC assembly assays to test the effect of the D52A mutation in RICE1 on removal of miRNA*s and nicked miRNA* in wheat germ. Flag-4Myc-AGO1, RICE1 wild type and D52A mutant protein were synthesized in wheat germ extract from TNT T7 Quick Coupled transcription/translation system before application of miR166/166* duplexes containing either 32P-labelled miR166 or miR166* (indicated by RED). The reaction mixtures were finally extracted with equal volumes of TE-saturated phenol. The RNA in the aqueous phase was recovered, analyzed with native 15–18% PAGE. The 32P signals were detected after exposure to a phosphorImager plate (Molecular Dynamics). Negative control reaction using mock-translated wheat germ was performed in parallel. Note: 9-nt and 12-nt miRNA* fragments were not detected in the red box region.