Figure 6. Increased processivity of AGO2-associated DICER1 in the presence of target mRNA.

(a) In vitro pre-miRNA processing assay with rAGO2 and rDICER1 reconfirmed target mRNA-driven increase in AGO2-associated DICER1 activity. In vitro pre-miRNA processing assay with rDICER1 and rAGO2 (native or heat-denatured) to quantify miR-122 biogenesis in the presence of target mRNA. Heat denaturation of rAGO2 was carried out at 95 °C for 5 min followed by rapid chilling. (b) In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and increasing concentrations of rAGO2 (10, 25 and 50 ng) in the presence of RL-con or RL-3 × bulge-let-7a (25 ng ml⁻¹). Mature let-7a levels were measured and plotted. (c) Schematic representation of RL-3 × bulge-let-7a_5BoxB mRNA used in the in vitro assays. In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and 50 ng rAGO2 in the presence of RL-3 × bulge-let-7a or RL-3 × bulge-let-7a_5BoxB mRNA (both at 25 ng μl⁻¹). Mature let-7a levels after the reaction were measured and plotted. (d) In vitro assay to measure the association of AGO2 and DICER1 along the 3′-UTR of target mRNAs. FH-AGO2 immunoprecipitated from HEK293 cells transiently expressing NHA-DICER1 was subjected to in vitro pre-miRNA processing assay with pre-miR-122 and RL-3 × bulge-miR-122 as described earlier, followed by immunoprecipitation of AGO2 and DICER1 with antibodies specific to endogenous proteins. Quantitative reverse transcriptase PCR (qRT-PCR) was done with indicated primers. (e) In vitro assay to measure processivity of DICER1. Immunopurified AGO2 (let-7a miRISC) incubated with 25 ng ml⁻¹ RL-con or RL-3 × bulge-let-7a in the presence of pre-miR-122 (10 nM) at 37 °C for 30 min followed by RNA isolation and quantification of mature miR-122 formed by qRT-PCR. Paired two-tailed Student's t-tests were used for all comparisons. *P<0.05, **P<0.01 and ***P<0.001. In a–e, values are means from at least three biological replicates. Error bars represent s.d.