C-terminal dsRBD of Dicer-2 is important for high-fidelity 21-nt siRNA production by purified recombinant proteins in vitro. (A) Silver-staining of the purified recombinant proteins (28 ng) run on a SDS–PAGE gel. (B) In vitro dicing assay using the purified recombinant proteins. As long dsRNA, body-labeled 104-bp dsRNAs with 2-nt 3′ overhang and either 5′ monophosphate or 5′ hydroxyl were tested. As short dsRNA, 5′ labeled 30-bp dsRNAs with 2-nt 3′ overhang and either 5′ monophosphate or 5′ hydroxyl were tested. The other end of the 30-bp dsRNAs was blocked by two deoxynucleotides (Cenik et al. 2011). Representative gel images (left panels) and quantification of the signals (right panels) are shown. Data are mean ± SD for three independent trials. (C) Length distribution of siRNAs produced from 104-bp dsRNA with 2-nt 3′ overhang and 5′ monophosphate by recombinant Dicer-2 proteins in test tube revealed by high-throughput sequencing. Data for the wild-type and phosphate-binding pocket mutant are from Kandasamy and Fukunaga (2016).
