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. 2012 May;18(5):945–957. doi: 10.1261/rna.032938.112

FIGURE 5.

FIGURE 5.

Influence of hairpin length on entry into the Ago2-dependent miRNA pathway. (A) The pre-mir-451 hairpin was extended by adding base pairs distal to the mature 23-nt miR-451 product (dark green box); thus, the sequence of mature miR-451 is not expected to change across this panel of length variants. Also highlighted are the inferred 12-nt 3′ passenger strand resulting from Ago2 cleavage (yellow box) and the 3′ resected region (light green box) following Ago2 cleavage. The solid reference line depicts the location of Ago2-mediated passenger strand cleavage; the dotted reference line depicts the putative location of 21-nt Dicer cleavage. (B) Northern blot analysis of the depicted constructs in Ago−/− MEFs reconstituted with wild-type Ago2 (Ago2-WT), with catalytic dead Ago2 (Ago2-CD), or in Dicer−/− MEFs, using a mature miR-451-5p probe in the common sequence of the panel of length variants. The blots were stripped and reprobed with U6 as a loading control. The absence of matured species in Ago2-CD cells for WT, +1, +2, and +3 constructs indicates that they are processed predominantly by the Ago2-dependent pathway; this is substantiated by their processing in Dicer−/− cells. With the +4 variant, a modest amount of Ago2-independent product appears and is enhanced in the +5, +6, and +7 constructs. This product is shorter than the predominant species detected in companion Dicer−/− cells, indicating that they are generated by distinct nucleases. The +5 variant is capable of maturing via both Ago2-dependent and Dicer-dependent pathways. The +7 variant is strictly Dicer-dependent. (C) Sensor assays of the length variant panel in HeLa cells and Dicer−/− MEFs against a miR-451 luciferase sensor bearing four seed matches. The repression capacity of Ago2-dependent and Dicer-independent hairpins is relatively equivalent in HeLa cells, except for the +3 and +4 variants (which correlates with their lower efficiency of biogenesis, panel B). A subset of these hairpins is also active in Dicer−/− MEFs, with shorter hairpin lengths exhibiting enhanced activity.