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. 2011 Sep 30;30(21):4414–4422. doi: 10.1038/emboj.2011.359

Figure 5.

Figure 5

Ago2-mediated cleavage of the CDR1 antisense transcript. (A) Firefly luciferase reporters (pISO) with a perfect miR-671 target (P target), the endogenous CDR1 antisense target (AS target), a mismatched target (MM target) or no target were co-transfected with a Renilla luciferase expression vector (pcDNA3-RL) and miR-671 (pJEBB-671) or miR-769 (pJEBB-769). Relative luminescence represents the Firefly/Renilla ratio for pJEBB-671 relative to pJEBB-769, normalized to the no target control. (B) Northern blot with RNA from HEK293 cells co-transfected with empty vector (EV), antisense wild-type (AS2) or antisense with an miR-769 target-site (AS2mt) and pJEBB-671 or pJEBB-769. Transient AS expression was determined with an AS-specific probe (upper panel). The NeoR probe reflects transfection efficiency of the AS expression vector (second panel), the eGFP probe shows ectopic gene expression from the transfected miRNA vector (third panel) and 18S serves as loading control (lower panel). (C) Set-up as in (A) but with transient co-transfection of Ago2-wild-type (Ago2-wt) or Ago2-slicer mutant (Ago2-D669A) expression vectors (Supplementary Figure S7A). (D) Antisense-specific qRT–PCR on RNA from HEK293 cells transiently transfected with miR-671 along with EV, Ago2 wild-type (Ago2-wt), or Ago2-slicer mutant (Ago2-D669A) expression vectors (Supplementary Figure S7B). (E) Northern blot on RNA from HEK293 cells transfected with either AS2 or AS2mt, after siRNA-mediated Xrn1 knockdown, showing AS2 full-length and 3′ cleavage fragment. (F) 5′ RACE on RNA from (E) showing 5′ ends of 3′ cleavage fragment and full-length AS2. (G) Clonal sequencing of cleavage fragments obtained from 5′ RACE (***P<0.001).