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. 2013 May 9;41(13):6568–6576. doi: 10.1093/nar/gkt361

Figure 4.

Figure 4.

Mechanistic studies of the effects of dsRBPs on Dicer by swapping domain and linker between TRBP and PACT. (A) Construct design for TRBP and PACT chimeric proteins swapping the third domain (T12P3 and P12T3) and linker region between domain 1 and domain 2 (TRBP-PL and PACT-TL). (B) Dicer–T12P3 (D-T12P3) and Dicer–P12T3 (D-P12T3) produce highly similar product miRNAs as Dicer–TRBP (D/T) and Dicer–PACT (D/P), respectively, in processing pre-miR-200a. Dicer–P12T3 processes pre-siRNA (dsRNA W1) much less efficiently than Dicer and Dicer–TRBP as in Figure 2C, which mimics Dicer–PACT processing (Figure 2C). For pre-miR-200a processing gel, each reaction has been quenched after 60 min by adding 1.2 volume of 2-fold formamide dye. The following steps were as same as in Figures 2A and 3A. For kinetic graphs, pre-let-7a, pre-miR-34c and dsRNA W1 were processed by Dicer–P12T3 in a single turnover condition with 10-fold excess amount of protein complex. [RNA] = 5 nM and [Dicer–P12T3] = 50 nM were incubated in the reaction buffer and the reaction was quenched at each time point by adding 1.2 volume of 2-fold formamide dye. The following steps were the same as described in Figures 2C and 3B. (C) Dicer–TRBP-PL and Dicer–PACT-TL process pre-miR-200a producing similar isomiR products to Dicer–TRBP and Dicer–PACT, respectively. Bar graphs show relative isomiR (1nt longer miR)/miR ratio in pre-miR-200a processing by Dicer–dsRBPs (Dicer–TRBP and Dicer–PACT) and Dicer-chimeric dsRBPs (Dicer–T12P3, Dicer–P12T3, Dicer–TRBP-PL and Dicer–PACT-TL). Error bar represents Standard Deviation (SD). (D) Dicer–PACT-TL processes pre-siRNA (dsRNA W1) much less efficiently than Dicer–TRBP-PL, while both Dicer–PACT-TL and Dicer–TRBP-PL processes pre-let-7a and pre-miR-34c efficiently. pre-let-7a, pre-miR-34c and dsRNA W1 were processed by Dicer–PACT-TL and Dicer–TRBP-PL in a single turnover condition with 10-fold excess amount of protein complex. [RNA] = 5 nM and [Dicer–TRBP-PL]/ [Dicer–PACT-TL] = 50 nM were incubated in the reaction buffer and the reaction was quenched at each time point by adding 1.2 volume of 2-fold formamide dye. The following steps were the same as described in Figures 2C and 3B.