FIGURE 5.

Slicer activity is activated on release of guide RNA from the Argonaute N-PAZ lobe. A, cleavage assay with DmAgo1 and DmAgo2. Argonaute protein is preincubated with bantam and added to radiolabeled target RNA that contains either a complete (perfect) or a partial (natural) complementary bantam binding site. Time-dependent accumulation of cleaved product was observed only for target with perfect match. B, cleavage assay with DmAgo1 conducted on targets with 12 consecutive base pairs across the bantam miRNA: 2–13 to 9–20 bp. A target with no base pairing served as a negative control (CTR, sense to bantam). A target RNA with perfect bantam site (1–23 bp, antisense to bantam) served as a positive control. C, RNA slicing activity of DmAgo1 was assessed for targets having different extents of complementarity: from 1–12 to 1–19 bp, and, finally, a complete match (1–23 bp). Cleavage products were observed when base pairing exceeded 15 nt, and cleavage activity was fully activated when pairing reached 17 nt. D, DmAgo1 variants in complex with bantam were added to target RNAs containing 1–13 through 1–16 pairings with bantam. DmAgo1-ΔN-PAZ showed enhanced slicer activity on 1–16 target, which was marginally cleaved by DmAgo1 or DmAgo1-PAZ6. E, potential base pairing between bantam and natural targets. F, extent of cleavage of natural targets with DmAgo1 and variants.